Abstract
Objectives
To establish the optimal parameters for group testing of pooled specimens for the detection of SARS-CoV-2.
Methods
The most efficient pool size was determined to be five specimens ...using a web-based application. From this analysis, 25 experimental pools were created using 50 µL from one SARS-CoV-2 positive nasopharyngeal specimen mixed with 4 negative patient specimens (50 µL each) for a total volume of 250 µL. Viral RNA was subsequently extracted from each pool and tested using the CDC SARS-CoV-2 RT-PCR assay. Positive pools were consequently split into individual specimens and tested by extraction and PCR. This method was also tested on an unselected group of 60 nasopharyngeal specimens grouped into 12 pools.
Results
All 25 pools were positive with cycle threshold (Ct) values within 0 and 5.03 Ct of the original individual specimens. The analysis of 60 specimens determined that 2 pools were positive followed by identification of 2 individual specimens among the 60 tested. This testing was accomplished while using 22 extractions/PCR tests, a savings of 38 reactions.
Conclusions
When the incidence rate of SARS-CoV-2 infection is 10% or less, group testing will result in the saving of reagents and personnel time with an overall increase in testing capability of at least 69%.
The B.1.1.529 (Omicron) variant of SARS-CoV-2 (the virus that causes COVID-19) was first detected in specimens collected on November 11, 2021, in Botswana and on November 14 in South Africa;* the ...first confirmed case of Omicron in the United States was identified in California on December 1, 2021 (1). On November 29, the Nebraska Department of Health and Human Services was notified of six probable cases
of COVID-19 in one household, including one case in a man aged 48 years (the index patient) who had recently returned from Nigeria. Given the patient's travel history, Omicron infection was suspected. Specimens from all six persons in the household tested positive for SARS-CoV-2 by reverse transcription-polymerase chain reaction (RT-PCR) testing on December 1, and the following day genomic sequencing by the Nebraska Public Health Laboratory identified an identical Omicron genotype from each specimen (Figure). Phylogenetic analysis was conducted to determine if this cluster represented an independent introduction of Omicron into the United States, and a detailed epidemiologic investigation was conducted. This activity was reviewed by CDC and was conducted consistent with applicable federal law and CDC policy.
.
Objectives:
During June–July 2021, an outbreak of SARS-CoV-2 occurred among attendees of a summer youth camp in Nebraska. We assessed the factors that contributed to onward transmission of disease.
...Methods:
The Four Corners Health Department conducted an outbreak investigation and recorded both laboratory-confirmed and self-reported cases of SARS-CoV-2 and mitigation measures employed. We generated sequences on positive specimens, created an epidemic curve to assist with outbreak visualization, and examined epidemiologic, genomic, and laboratory outcomes.
Results:
Evaluation of 3 index cases led to the identification of 25 people with COVID-19 who interacted directly with the camp. Contact tracing revealed an additional 18 cases consistent with onward community transmission. Most (24 of 35, 68.5%) vaccine-eligible community cases were not vaccinated. We sequenced 8 positive specimens; all were identified as the Delta variant. Precamp planning incorporated local health officials who recommended wearing face masks, practicing social distancing, and using attendee cohorts to limit mixing of people involved in various activities.
Conclusion:
Low vaccination levels and poor face mask–wearing habits among attendees resulted in secondary and tertiary spread of SARS-CoV-2 and severe outcomes among young adults. This outbreak of COVID-19 at a youth camp highlights the importance of vaccination and use of other measures to interrupt opportunities for SARS-CoV-2 spread in the community and shows that vaccinated people remain vulnerable to infection when in an environment of high exposure to SARS-CoV-2. Proactive case identification and interruption of chains of transmission can help decrease the number of cases and avoid further severe outcomes.
Clostridioides difficile infection (CDI) has become a threatening public health problem in the developed world. In the kingdom of Saudi Arabia, prevalence of CDI is still unknown due to limited ...surveillance protocols and diagnostic resources. We used a two-step procedure to study and confirm C. difficile cases. We also studied toxin profiles of these isolates.
Stool samples were collected from symptomatic patients and clinically suspected of CDI for almost 12 months. Isolates were confirmed by culture method followed by 16S rRNA sequencing. Multiplex PCR was performed for the identification of toxin A, toxin B and binary toxin genes and compared to Gene Expert results.
Out of the 47 collected samples, 27 were successfully grown on culture media. 18 samples were confirmed as C. difficile by both culture and 16S rRNA sequencing. Interestingly, the rest of the isolates (9 species) belonged to different genera. Our results showed 95% of samples were positive for both toxin A and B (tcdA, tcdB) and all samples exhibited the toxin gene regulator tcdC. All samples were confirmed negative for the binary toxin gene ctdB and 11% of the isolates were positive for ctdA gene. Interestingly, one isolate harbored the binary toxin gene (cdtA+) and tested negative for both toxins A and B.
We believe that combining the standard culture method with molecular techniques can make the detection of C. difficile more accurate.
is an emerging multi-drug resistant pathogen with high mortality rate; nosocomial infections have been reported worldwide, causing a major challenge for clinicians and microbiological laboratories. ...The study aims to describe new cases of
and detect drug resistance-associated mutations of
by the sequencing of
and
genes. A total of six specimens were collected from blood, urine, ear swab, and groin screening samples. Isolates were incubated for 48 h on Sabouraud Dextrose agar (SDA) at 42 °C, then confirmed by MALDI-TOF MS. Furthermore, antifungal susceptibility testing was performed using the Vitek 2 system to detect Minimum Inhibitory Concentrations (MICs) of six antifungals. Sequences of
gene and ITS regions from isolates and phylogenetic analysis were performed. Gene sequencing was analysed to detect drug resistance-associated mutations by
and
genes sequencing. All
isolates were confirmed by MALDI-TOF MS, and evolutionary analyses using sequences of
gene and ITS region. Antifungal susceptibility testing showed that all isolates were resistant to fluconazole. Sequencing of
and
genes from the isolates revealed the presence of two (F132Y and K143R) drug resistance-associated mutations in
, however,
gene was devoid of mutations. The study sheds light on a public health threat of an emerging pathogen, and the hospital implemented strict contact screening and infection control precautions to prevent
infection. Finally, there is a critical need to monitor the antifungal resistance in different geographical areas and implementation of efficient guidelines for treatment.
Strongyloides stercoralis is an uncommon infection in Saudi Arabia. It can establish latency and cause an autoinfection in humans that lasts for years. The infection can get reactivated during ...immunosuppression and can result in a life-threatening Strongyloides hyperinfection syndrome. We present three cases of renal transplant recipients who developed Strongyloides infection following transplantation. A bronchoalveolar lavage specimen, a duodenal biopsy and/or a stool specimen from these patients revealed evidence of S. stercoralis larvae. The first two patients received kidneys from the same deceased donor, a native of Bangladesh, an area that is highly endemic for S. stercoralis. The data suggest that the first two cases might be donor derived. High-risk donors and recipients should be screened for Strongyloides infection to initiate treatment before transplantation thus reducing morbidity and mortality.
Background:
In late September 2021, a cluster of patients with nosocomial COVID-19 was identified on a liver transplant unit at University of Nebraska Medical Center.
Methods:
The outbreak ...investigation included contact tracing via patient chart and employee health record reviews and serial prevalence testing for SARS-CoV-2 among potentially exposed patients and healthcare workers (HCWs). Routine admission and preprocedural screening for SARS-CoV-2 was performed, and involved patients had negative admission screening results with positive SARS-CoV-2 tests >5 days from admission. Mitigation strategies involved reinforcement of patient care and visitation procedures. Whole-genome sequencing of positive SARS-CoV-2 specimens was conducted.
Results:
The potential outbreak cluster included 6 patients in the same quadrant of the liver transplant unit, 1 visitor, and 11 healthcare workers (Fig. 1). Moreover, 4 patients had severe liver disease, including 2 with liver transplants. All HCWs and half of the patients had received 2 doses of mRNA vaccine, albeit >5 months from their second vaccination. Whole-genome sequencing confirmed patients 1–6 and HCWs 1–3 had related transmission of COVID-19. However, infections in HCWs 4–6, who worked in a transplant-related office setting without patient contact, were due to 2 separate introductions of SARS-CoV-2 unrelated to the hospital outbreak. Sequencing could not be performed on HCWs 7–11 due to low viral concentration in the original specimens or unavailable specimen. The SARS-CoV-2 δ (delta) variant (B.1.617.2) was identified in all sequenced samples. HCWs 8–10 were asymptomatic and had had contact with each other and had been involved with an intubation without proper PPE for SARS-CoV-2 on patient 6. HCW 8 had had contact with all 6 patients and HCW 9 had had contact with 5 patients. A clear index case could not be identified; however, we suspect that the index case was either visitor 1, who tested positive during patient 2’s admission, or an asymptomatic healthcare worker (HCWs 8–10).
Conclusions:
We identified a nosocomial outbreak of the SARS-CoV-2 δ (delta) variant in a solid-organ transplant unit including patients, a visitor, and vaccinated healthcare workers with multiple introductions of the virus. Further transmission was not detected after enhanced infection control measures were introduced, including universal masking and eye protection, closing patient doors, and enforcement of visitor masking policy. We describe the difficulties tracing SARS-CoV-2 transmission in the hospital setting, even with advanced sequencing techniques. This outbreak highlights the importance of booster vaccination and strict infection control practices, especially in the setting of the δ (delta) variant.
Funding:
None
Disclosures:
None
Shiga-toxin producing Escherichia coli (STEC) O26:H11 is the second most common cause of severe diarrhea and hemolytic uremic syndrome worldwide. The implementation of whole genome sequencing (WGS) ...enhances the detection and in-depth characterization of these non-O157 STEC strains. The aim of this study was to compare WGS to phenotypic serotyping and pulse field gel electrophoresis (PFGE) for characterization of STECO26 strains following a zoonotic outbreak from cattle to humans.
This study evaluated seven E. coli strains; two strains isolated from two children with gastrointestinal symptoms and five strains from five calves suspected as the source of infection. Six of these isolates were serotyped phenotypically and by WGS as E. coli O26:H11 while one bovine isolate could be serotyped only by WGS as E. coli O182:H25. Stx1 was detected in two human- and two bovine-isolates using PCR and WGS. Using WGS, all four STECO26 isolates belong to sequence type (ST) 21 while the two stx1 negative E. coli O26 were ST29. All four STECO26 isolates were indistinguishable by PFGE. However, the data generated by WGS linked the two human STECO26 isolates to only one bovine STECO26 strain by having identical high-quality single nucleotide polymorphisms (hqSNPs) and identical virulence factor profiles while the remaining bovine STECO26 isolate differed by 7 hqSNPs and lacked virulence factor toxB.
These data demonstrated that WGS provided significant information beyond traditional epidemiological tools allowing for comprehensive characterization of the STEC. Using this approach, WGS was able to identify the specific source of infection in this study.
A role for human cytomegalovirus (HCMV) in the pathogenesis of glioblastoma multiforme (GBM) was proposed more than a decade ago and has since generated a considerable debate as a possible ...therapeutic target. We investigate the presence of HCMV in the specimens of patients with GBM treated in our centre. This is a retrospective cohort study to investigate the presence of HCMV by routine immunohistochemical stains and polymerase chain reaction (PCR)-based molecular analysis on formalin-fixed-paraffin-embedded tissue of all patients with GBM treated in our hospital in 2009-2013 (5 years). The evaluation of positivity by immunohistochemistry (IHC) was semi-quantitative. The molecular analysis was performed by extracting the tumour DNA from representative paraffin-embedded tissue blocks and amplified for detection by a sensitive real time PCR (RT-PCR) CMV assay. During the study period, we treated 45 patients with GBM; however, adequate pathology tissue materials were available only for 32 patients. All the pathology material was reviewed and the diagnosis was confirmed. All the cases were found to be negative for CMV expression by our IHC and RT-PCR CMV assay. Our study has shown no expression of CMV in GBM. Our results were similar to other recent reports that concluded insufficient evidence to recommend routine testing for CMV in GBM or treatment as an add-on therapy.
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