Hypervirulent Capsular Serotypes K1 and K2 Klebsiella pneumoniae Strains Demonstrate Resistance to Serum Bactericidal Activity and Galleria mellonella Lethality.

Hypervirulent Klebsiella pneumoniae (hvKp) is a variant that has been increasingly linked to severe, life-threatening infections including pyogenic liver abscess and bloodstream infections. HvKps belonging to the capsular serotypes K1 and K2 have been reported worldwide, however, very scarce studies are available on their genomics and virulence. In the current study, we report four hypermucoviscous extended-spectrum ß-lactamase-producing hvKp clinical strains of capsular serotype K1 and K2 isolated from pus and urine of critically ill patients in tertiary care hospitals in Oman. These strains belong to diverse sequence types (STs), namely ST-23(K1), ST-231(K2), ST-881(K2), and ST-14(K2). To study their virulence, a Galleria mellonella model and resistance to human serum killing were used. The G. mellonella model revealed that the K1/ST-23 isolate was the most virulent, as 50% of the larvae died in the first day, followed by isolate K2/ST-231 and K2/ST-14, for which 75% and 50% of the larvae died in the second day, respectively. Resistance to human serum killing showed there was complete inhibition of bacterial growth of all four isolates by the end of the first hour and up to the third hour. Whole genome sequencing (WGS) revealed that hvKp strains display a unique genetic arrangement of k-loci. Whole-genome single-nucleotide polymorphism-based phylogenetic analysis revealed that these hvKp isolates were phylogenetically distinct, belonging to diverse clades, and belonged to different STs in comparison to global isolates. For ST-23(K1), ST-231(K2), ST-881(K2), and ST-14(K2), there was a gradual decrease in the number of colonies up to the second to third hour, which indicates neutralization of bacterial cells by the serum components. However, this was followed by a sudden increase of bacterial growth, indicating possible resistance of bacteria against human serum bactericidal activity. This is the first report from Oman detailing the WGS of hvKp clinical isolates and assessing their resistance and virulence genomics, which reinforce our understanding of their epidemiology and dissemination in clinical settings.

External quality assessment of laboratory performance in bacteriology in the Eastern Mediterranean Region, 2011-2019.

Background: Since 2007, national public health laboratories in the WHO Eastern Mediterranean Region (EMR) have participated in a regional external quality assessment scheme in bacteriology to improve testing proficiency. Aims: To assess laboratory performance in bacteriology in the EMR between 2011 and 2019 using the regional external quality assessment scheme. Methods: We analysed the accuracy of participant-reported data in bacterial identification, Gram stain microscopy, and antimicrobial susceptibility testing. For each category, we assessed the performance over time, the performance on multiple organisms, and whether a laboratory repeatedly failed to attain satisfactory results. Results: Between 2011 and 2019, 70% of laboratories achieved satisfactory performance for bacterial identification and antimicrobial susceptibility testing, and 85% performed satisfactory Gram stain microscopy. Testing did not improve on multiple organisms and results were consistently low for some pathogens and test categories. Twenty-nine percent of laboratories underperformed throughout the study period. Conclusion: The unchanged performance over time and underperformance of laboratories highlight the need for improvements in the regional external quality assessment scheme. Participating laboratories and WHO need to work more actively to strengthen the problem areas.

The First Confirmed Pediatric Chronic Osteomyelitis due to Coxiella Burnetii in Oman.

We describe here the first confirmed case in Oman of chronic osteomyelitis due to Coxiella burnetii, in a previously healthy four-year-old Omani girl. After laboratory confirmation of C. burnetii infection using molecular and qualitative and quantitative serological assays, the case was successfully managed with a combination of oral ciprofloxacin and cotrimoxazole and thereafter followed up for a long period without remission.

Genomic analysis of the emerging carbapenem-resistant Klebsiella pneumoniae sequence type 11 harbouring Klebsiella pneumoniae carbapenemase (KPC) in Oman.

BACKGROUND AND OBJECTIVE: The presence of carbapenemase enzymes among Enterobacterales is the main mechanism to reduce susceptibility to a wide range of antibiotics. Carbapenemase enzymes such as the Klebsiella pneumoniae carbapenemase (KPC) hydrolyse beta-lactam antibiotics group, which includes carbapenem, leads to fewer treatment options. We aim to describe the first report of carbapenem-resistant K. pneumoniae (CRKP) sequence type (ST) 11 harbouring KPC in Oman. MATERIAL AND METHODS: Five confirmed CRKP isolates were isolated from clinical samples during the period of January 2019 till December 2019. Strains were genotyped by pulse field gel electrophoresis (PFGE) for genetic relatedness. Whole genome sequencing (WGS) was performed to observe relationships with global strains using multilocus sequence typing (MLST). Antimicrobial genes, capsular loci-K-types, plasmids types and virulence genes were also identified using whole genome sequence data. RESULTS: All five CRKP were determined to have blaKPC-2 with or without blaOX-A48 and blaNDM-2. The molecular genotyping by PFGE showed 100% similarity among the five isolates. The MLST allelic profile analysis clonally clustered our strains with SL-258, CG-11 and ST11 mainly reported from South Asia. Further molecular characterization of the capsular K-locus and O-locus genes, revealed the strains to belong to KL-47 type and OL101 type respectively. The core genome typing suggests that our strains were clonally related to Chinese strains with less than five chromosomal nucleotides differences. CONCLUSION: Epidemiological and molecular analyses confirmed that these KPC-producing K. pneumoniae strains are from a single clone that caused multiple nosocomial infections in one health institution. This finding highlights the importance to sustain the surveillance and infection prevention efforts and to step up active screening to prevent the spread of nosocomial infection.

Laboratory Performance and Clinical Correlation of Real-time Polymerase Chain Reaction as a Diagnostic Test for Bordetella Pertussis Isolated from Patients in Oman.

Objectives: To evaluate real-time polymerase chain reaction (PCR) as a diagnostic tool for pertussis (whooping cough), vis-à-vis culture, the long-time gold standard, with emphasis on the importance of clinical correlation in determining specificity. Methods: Nasopharyngeal/throat swab samples in charcoal media received from all over Oman at Central Public Health Laboratories from January 2014 to December 2016 were included in this study. These samples were tested using both culture and real-time PCR. PCR was compared with culture to calculate its sensitivity and specificity. Further clinical correlation was conducted for the discrepant cases using different case definitions. Results: A total of 590 nasopharyngeal/throat samples were included in the study. Out of the 590 samples, 73 were positive by PCR compared with 26 positive samples by culture (which were also positive by PCR). The sensitivity and specificity of PCR compared with those of culture were 100% (95% CI: 86.77-100) and 91.7% (95% CI: 89.07-93.81), respectively. To rule out false-positive results by PCR, clinical correlation was performed. Out of 47 cases that were positive by PCR but negative by culture, 44 cases were clinically evaluated by accessing clinical details. Out of these 44 cases, 21 (47.7%) met the pertussis clinical criteria according to the CDC-2014 case definition, 41 (93.2%) according to the Europe-2008 case definition, and 44 (100%) according to the Canada-2009 and Australia-2014 case definitions. With only positive PCR, these cases were classified as confirmed according to these case definitions, which increased the specificity of PCR to 95.7-100%. The mean turnaround time was 3.5 days for PCR compared to 6.2 days for culture. Conclusions: Real-time PCR is a highly sensitive and specific test for the diagnosis of Bordetella pertussis infection. Based on our results, we recommend setting up PCR diagnostic facilities in regional hospitals in Oman as this will lead to a timely and accurate diagnosis of pertussis.

Burkholderia pseudomallei Septic Arthritis of the Knee Joint: Report of a Third Imported Case in Oman.

Melioidosis is a severe disease that can affect humans and animals. It is caused by Burkholderia pseudomallei, which is an environmental aerobic gram negative bacteria. Despite being endemic in Southeast Asia and Northern Australia, many cases have been reported from different regions, including the Middle East. This is the third imported case of B. pseudomallei from Oman in a 46-year-old Indian male with left knee septic arthritis in less than three weeks after arrival in Oman. He underwent open arthrotomy, and his synovial fluid culture grew a bacteria with dry, pink colonies that were oxidase positive, susceptible to amoxicillin-clavulanic, identified by API NE as B. pseudomallei, and confirmed by molecular tests. This single case report highlights the urgent need to increase molecular diagnostic capacity and improve public health surveillance while maintaining a one health tripartite approach.

Evaluation of Four Rapid Antigen Tests for Detection of SARS-CoV-2 virus.

OBJECTIVES: Considering the increasing, significant burden that coronavirus disease 2019 (COVID-19) imposes on the healthcare system, the need for simple, rapid, and affordable diagnostic tests to support the existing costly and demanding polymerase chain reaction (PCR) assay becomes required. This prospective diagnostic test accuracy study aims to evaluate the performance of four different COVID-19 rapid antigen tests compared to real-time reverse transcription PCR (rRT-PCR) between June and July 2020 to determine the feasibility of integrating these tests into the diagnostic algorithm in clinical settings. METHODS: Swabs were collected from 306 patients and analyzed using rRT-PCR and antigen tests from four different providers. RESULTS: The antigen tests' sensitivities were 65.8%, 69.8%, 64.0%, and 64.3% for the STANDARD™ Q COVID-19 Ag test, PCL COVID-19 Ag Rapid fluorescent immunoassay (FIA) test, BIOCREDIT COVID-19 Ag test, and Sofia SARS-CoV-2 antigen FIA test, respectively. Specificity was 94.1% for PCL COVID-19 Ag Rapid test and 100% for the other three assays. All assays showed a significant negative correlation between the reference rRT-PCR Ct values and Ag test results. Besides, sensitivities of the STANDARD™ Q COVID-19 Ag test, PCL COVID-19 Ag Rapid FIA test, and BIOCREDIT COVID-19 Ag test improved to ≥ 85% after exclusion of samples with PCR Ct values > 30. CONCLUSIONS: The high specificity of the rapid antigen tests and other parameters like simplicity, rapidity, and affordability suggest that antigen tests are likely to be helpful if integrated and interpreted appropriately in stepwise diagnostic algorithms. Given the low sensitivity of 64.0-69.8% of the antigen tests, we recommend that clinically relevant negative results undergo further testing Ag to confirm or exclude a COVID-19 diagnosis.

Validation of GeneXpert testing for SARS-CoV-2 RNA in eight hospital laboratories in Oman.

In response to the current COVID-19 pandemic, numerous commercial assays have been developed for the detection of SARS-CoV-2 for use in the clinical diagnostic laboratories. To date, there is limited comparison of testing methods performed in different hospital laboratory sites. The aims of the study were to evaluate the analytical performance of Cepheid Xpert Xpress SARS-CoV-2 when compared to RT-PCR. This is a cross-sectional study. A total of 155 nasopharyngeal swabs were taken in duplicate from patients presenting with suspected COVID-19 to 8 hospitals in Oman. One swab was tested by the hospital laboratory and the duplicate swab was sent to the national Central Public Health Laboratory (CPHL) for testing. We compared the analytical performance of the commercially available point of care Cepheid Xpert Xpress SARS-CoV-2 assay which was used in the 8 different hospitals with assays including Liferiver, Sansure, TIB MOL BIOL, Kingfisher and COBAS 6800 by Roche which were performed at the CPHL. Testing of the duplicate swabs revealed excellent agreement of results with the viral loads of Ct values ranging from 16-43 for the E gene, 18-44 for the N gene and 17-44 for the ORF gene using the Liferiver assay. The overall sample sensitivity and specificity of the Cepheid Xpert Xpress SARS-CoV-2 assay were both 100% and there was 100% agreement across specimens. We conclude that the rapid GeneXpert and RT-PCR kits assessed in this study may be used for routine diagnostic testing of COVID-19 patients by experienced clinical microbiology diagnostic laboratories. Our results highlight the importance of rapid molecular testing at different sites within a country in a public health emergency.

Seroprevalence of SARS-CoV-2 antibodies in the general population of Oman: results from four successive nationwide sero-epidemiological surveys.

OBJECTIVE: To assess the seroprevalence of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in Oman and longitudinal changes in antibody levels over time within the first 11 months of the coronavirus disease 2019 (COVID-19) pandemic. METHODS: This nationwide cross-sectional study was conducted as a four-cycle serosurvey using a multi-stage stratified sampling method from July to November 2020. A questionnaire was used and included demographics, history of acute respiratory infection and list of symptoms, COVID-19 contact, previous diagnosis or admission, travel history and risk factors. RESULTS: In total, 17,457 participants were surveyed. Thirty percent were female and 66.3% were Omani. There was a significant increase in seroprevalence throughout the study cycles, from 5.5% (4.8-6.2%) in Cycle 1 to 22% (19.6-24.6%) in Cycle 4. There was no difference in seroprevalence between genders, but significant differences were found between age groups. There was a transition of seroprevalence from being higher in non-Omanis than Omanis in Cycle 1 [9.1% (7.6-10.9%) vs 3.2% (2.6-3.9%)] to being higher in Omanis than non-Omanis in Cycle 4 [24.3% (21.0-27.9%) vs 16.8% (14.9-18.9%)]. There was remarkable variation in the seroprevalence of SARS-CoV-2 according to governorate. Close contacts of people with COVID-19 had a 96% higher risk of having the disease [adjusted odds ratio (AOR) 1.96, 95% confidence intervals (CI) 1.64-2.34]. Labourers had 58% higher risk of infection compared with office workers (AOR 1.58, 95% CI 1.04-2.35). CONCLUSION: This study showed a wide variation in the spread of SARS-CoV-2 across governorates in Oman, with higher estimated seroprevalence in migrants in the first two cycles. Prevalence estimates remain low and are insufficient to provide herd immunity.

Community Foodborne of Salmonella Weltevreden Outbreak at Northern Governorate, Sultanate of Oman.

OBJECTIVES: To investigate the course of a community gastroenteritis outbreak by Salmonella and implement interventional activities and roles to prevent occurring such an outbreak in the future. METHODS: From August 27 to 2 September 2015, 101 individuals were reported among a local community. All affected individuals had a history of food consumption at a local restaurant. A rapid response team conducted active surveillance and interview with the affected individuals and workers of the restaurant. Food items and stools from food handlers and affected individuals were cultured and sent for genotyping. An environmental audit of the restaurant had been conducted. RESULTS: The total majority of the affected individuals were male and more than 70% belonged to the young age group from 15 to 45 years. Out of the total, 97% had diarrhea, 70% fever, 56% abdominal cramps and 49% vomiting. All those affected were managed symptomatically except for 14 cases admitted for intravenous rehydration. Breakdown of food safety and basic personal hygiene were detected in the environment of the restaurant and among the workers. There are 39 out of 49 stool cultures of cases, six out of 18 food handlers, and five food samples were positive for Salmonella spp. The identical DNA fingerprinting pattern among S. Weltevreden strains originating from human cases and food was detected. CONCLUSION: This is the first reported community foodborne of S. Weltevreden outbreak in Oman. The importance of food safety and rigors environmental safety is emphasized. Basic personal hygiene and training of food handlers in restaurants are recommended with public health measurements.

Characteristics, Risk Factors, and Survival Analysis of Candida auris Cases: Results of One-Year National Surveillance Data from Oman.

BACKGROUND: Candida auris (C. auris) is an emerging healthcare-associated pathogen resulting in significant morbidity and mortality. The aim of this study is to report data from the national C. auris surveillance system for 2019 and conduct a survival analysis of the reported cohort. METHODS: a retrospective analysis was conducted for all C. auris cases reported nationally to the Oman Antimicrobial Surveillance System (OMASS) in 2019, and isolates were sent to the Central Public Health Laboratories (CPHL). Clinical and demographic data were obtained through the E-Surveillance reporting system and the Electronic System (NEHR Al-Shifa) at CPHL. Statistical analysis was done using Kaplan-Meier analysis and Cox proportional hazard models. RESULTS: One hundred and twenty-nine isolates of C. auris were grown from 108 inpatients; 87% were isolated from clinical samples, of which blood was the most common (38.9%). Forty (37%) were ≥65 years of age, 72 (66.7%) were males, and 85 (78.7%) were Omani nationals. Of the total isolates, 43.5% were considered as colonization; 56.5% were considered infection, of which 61.8% of them were candidemia. At least one risk factor was present in 98.1% of patients. The mean time from admission to infection was 1.7 months (SD = 2.8), and the mean length of hospital stay was 3.5 months (SD = 4). Totals of 94.8% and 96.1% of the isolates were non-susceptible to fluconazole and amphotericin, respectively. The variables found to be significantly associated with longer survival post C. auris diagnosis (p < 0.05) were age < 65 years, absence of comorbidities, length of stay < 3 months, colonization, and absence of candidemia. The infection fatality rate was 52.5%. CONCLUSION: Including C. auris in an ongoing antimicrobial surveillance program provides important data for the comprehensive management of this growing public health threat. The current study shows health care outbreaks of C. auris are ongoing, with 52.5% infection fatality, although our isolates remained sensitive to Echinocandins in vitro.

Genomic analysis of the first cases of extensively drug-resistant, travel-related Salmonella enterica serovar Typhi in Oman.

Objectives: To highlight the importance of molecular testing in characterizing extensively drug-resistant (XDR) Salmonella Typhi (S. Typhi), and linking it to the current outbreak in Sindh, Pakistan. Methods: Our study reports three travel-related typhoid fever cases caused by XDR S. Typhi that presented between January 2019 and August 2019. Antimicrobial susceptibility and genotyping with pulse-field gel electrophoresis (PFGE) were carried out. Whole-genome sequencing (WGS) was performed to characterize the genomic clonality in relation to the emerging outbreak of S. Typhi in Sindh, Pakistan, and to study the molecular resistance profiles. Results: Laboratory testing revealed resistance to all first-line antibiotics (i.e ampicillin, chloramphenicol, and trimethoprim-sulfamethoxazole), as well as to quinolones and third-generation cephalosporins, leading to a change in the patients' therapy to the use of carbapenems. Classical MLST (cMLST) revealed that the strains were of sequence type 1 (ST1) and the core genome sequence (cgWGS) analysis closely clustered our strains with internationally reported strains from Pakistan, India, and the UK. The strains were found to carry a bla CTX-15 gene-harbouring IncY plasmid, which encodes resistance to ceftriaxone. Conclusions: Our report alerts clinicians to the use of appropriate empirical treatments in such scenarios, and highlights the significance of the global spread of XDR S. Typhi.

Ongoing Challenges with Healthcare-Associated Candida auris Outbreaks in Oman.

Candida auris has emerged in the past decade as a multi-drug resistant public health threat causing health care outbreaks. Here we report epidemiological, clinical, and microbiological investigations of a C. auris outbreak in a regional Omani hospital between April 2018 and April 2019. The outbreak started in the intensive care areas (intensive care unit (ICU), coronary care unit (CCU), and high dependency unit) but cases were subsequently diagnosed in other medical and surgical units. In addition to the patients' clinical and screening samples, environmental swabs from high touch areas and from the hands of 35 staff were collected. All the positive samples from patients and environmental screening were confirmed using MALDI-TOF, and additional ITS-rDNA sequencing was done for ten clinical and two environmental isolates. There were 32 patients positive for C. auris of which 14 (43.8%) had urinary tract infection, 11 (34.4%) had candidemia, and 7 (21.8%) had asymptomatic skin colonization. The median age was 64 years (14-88) with 17 (53.1%) male and 15 (46.9%) female patients. Prior to diagnosis, 21 (65.6%) had been admitted to the intensive care unit, and 11 (34.4%) had been nursed in medical or surgical wards. The crude mortality rate in our patient's cohort was 53.1. Two swabs collected from a ventilator in two different beds in the ICU were positive for C. auris. None of the health care worker samples were positive. Molecular typing showed that clinical and environmental isolates were genetically similar and all belonged to the South Asian C. auris clade I. Most isolates had non-susceptible fluconazole (100%) and amphotericin B (33%) minimal inhibitory concentrations (MICs), but had low echinocandin and voriconazole MICs. Despite multimodal infection prevention and control measures, new cases continued to appear, challenging all the containment efforts.

Serotype distribution and antibiotic resistance among invasive Streptococcus pneumoniae from Oman post 13-valent vaccine introduction.

OBJECTIVE: This study was undertaken to determine the serotype distribution and drug susceptibility patterns in pneumococcal isolates recovered from patients with invasive pneumococcal disease (IPD). METHODS: All invasive pneumococcal isolates received between June 2014 and June 2016 were included in the study as part of a national laboratory-based IPD surveillance program. Isolates recovered from clinical specimens of normally sterile body sites were included. RESULTS: A total of 41 different serotypes were identified among the 132 pneumococcal isolates included in this study. The most prevalent serotypes/serogroups were 12 (8.3%), 15 (8.3%), 19F (7.6%), 3 (6.1%), and 19A (6.1%);. It was observed that overall vaccine coverage rates for pneumococcal conjugate vaccines (PCV), PCV7, PCV10 and PCV13 were 15.9%, 24.2% and 37.1% respectively. 56.8% (n=75) of the isolates were non-susceptible to at least one antibiotic and 40.9% (n=54) of the isolates were resistant to PEN (M). 18.9% (n=25) of the isolates were multi-drug resistant (MDR).The case fatality rate was 15.9%. CONCLUSION: Our study results call for broader vaccine coverage, emphasizes the need to introduce the conjugate pneumococcal vaccine for the high risk adult population and stress the importance of continuous surveillance of serotypes and antimicrobial resistance to guide vaccine development and antimicrobial stewardship activities.