Evaluation of Immulex S. pneumoniae Omni test for the direct detection of S. pneumoniae from positive blood cultures.

Rapid and early identification of Streptococcus pneumoniae from positive blood cultures is crucial for the management of patients with bloodstream infections (BSI). Many identification systems in microbiology laboratories have difficulty differentiating S. pneumoniae from other closely related species in the Streptococcus mitis group. To overcome this limitation, we developed a rapid workflow in our laboratory combining direct MALDI-TOF MS identification with the Immulex S. pneumoniae Omni test (SSI Diagnostica, Denmark) for rapid detection of S. pneumoniae directly from positive blood cultures. The workflow was evaluated using 51 Streptococcus isolates. Compared to conventional biochemical testing, our new workflow demonstrates 100 % specificity and sensitivity for the detection and differentiation of S. pneumoniae from other closely related species. Our new workflow is accurate, cost-effective, and can easily be implemented in microbiology laboratories that already perform direct MALDI-TOF identification from positive blood cultures to improve the management of patients with invasive pneumococcal disease. Importance: Invasive pneumococcal disease remains a major public health problem worldwide. Reducing the time to identify Streptococcus pneumoniae in positive blood cultures allows patients to be treated sooner with more targeted and effective antibiotics. We evaluated a two-step protocol where positive blood cultures are first tested directly by MALDI-TOF MS and any samples containing Streptococcus species are tested by Immulex S. pneumoniae Omni test to both detect and differentiate S. pneumoniae from other closely related Streptococcus species. Our study results showed 100 % sensitivity and specificity, and a much faster turn-around time than conventional methods.

Monitoring the effect of environmental conditions on safety of fresh produce sold in Qatar's wholesale market.

Fresh produce imported by Qatar are mostly sold at the wholesale produce market (WPM) located in open-air and near major animal markets and slaughterhouses. This study was the first in Qatar to monitor the effect of environmental conditions on the microbial quality and safety of fresh produce sold at the WPM over 1 year. The monitoring involved the collection of 540 produce samples along with samples of air, soil, and surface swabs. Samples were analyzed for total aerobic bacteria (TAB); generic Listeria spp., Staphylococcus spp., Salmonella spp.; total coliforms and total fungi. Bacterial and fungal isolates were identified using 16S rRNA/ITS rRNA markers. Environmental/sanitary factors significantly impacted the prevalence of microorganisms in all samples tested. Produce quality was rated 'poor' during the months of November-February or May-August, with TAB and coliform counts exceeding 6 and 4 log10 CFU/g, respectively. Bacillus subtilus, Enterobacter cloacae, E. faecium, P. expansium, P. aurantiocandidum, and A. niger were the most abundant species with prevalence rate of 11-30%. The high microbial load of environmental samples indicates that the location of the WPM near livestock markets is likely impacting the microbial quality of fresh produce. Therefore, effective control measures need to be implemented at WPM to improve produce safety yearlong.

Evaluation of Rapid Immunochromatographic Tests for the Direct Detection of Extended Spectrum Beta-Lactamases and Carbapenemases in Enterobacterales Isolated from Positive Blood Cultures.

NG-Test CTX-M MULTI and NG-Test Carba 5 (NG Biotech) are two rapid in vitro immunochromatographic assays that are widely used for the detection of the most common extended spectrum beta-lactamases (ESBL) and carbapenemases in Enterobacterales. ESBL and carbapenemases are leading causes of morbidity and mortality worldwide and their rapid detection from positive blood cultures is crucial for early initiation of effective antimicrobial therapy in bloodstream infections (BSI) involving antibiotic-resistant organisms. In this study, we developed a rapid workflow for positive blood cultures for direct identification of Enterobacterales by MALDI-TOF mass-spectrometry, followed by detection of ESBL and carbapenemases using NG-Test CTX-M MULTI and NG-Test Carba 5 (NG Biotech). The workflow was evaluated using Enterobacterales isolates (n = 114), primarily Klebsiella species (n = 50) and Escherichia coli (n = 40). Compared to the standard testing approach in our institution using BD Phoenix, our new testing approach demonstrates 100% sensitivity and specificity for organism identification and detection of ESBL and carbapenemases. Implementation of a rapid workflow in diagnostic microbiology laboratories will enable more effective antimicrobial management of patients with BSI due to ESBL- and carbapenemase-producing Enterobacterales. IMPORTANCE The incidence of bloodstream infections (BSI) with extended spectrum beta-lactamase (ESBL) producing and carbapenemase producing Enterobacterales (CPE) is increasing at an alarming rate, for which only limited therapeutic options remain available. Rapid identification of these bacteria along with their antibiotic resistance mechanisms in positive blood cultures with Gram-negative bacteria will allow for early initiation of effective therapy and limit the overuse of broad-spectrum antibiotics in BSI (1). In this study we evaluated a combined approach of testing positive blood cultures directly, using MALDI-TOF MS followed by rapid immunochromatographic tests, for the detection of ESBLs and CPEs. Our approach demonstrates 100% sensitivity and specificity for the identification of Enterobacterales and detection of ESBLs and CPEs in positive blood culture with a turnaround time (TAT) of ≤60 min compared to a TAT of 48 h required by conventional culture and susceptibility testing methods.

Performance and Impact on Initial Antibiotic Choice of Direct Identification of Pathogens from Pediatric Blood Culture Bottles Using an In-House MALDI-TOF MS Protocol.

The performance and early therapeutic impact of direct identification by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF; DIMT) on pediatric blood culture bottles using in-house-developed methods to obtain microbial pellets for spectrometric analysis have seldom been studied. During a 2-year period (June 2018 to May 2020), DIMT was performed on broths from positive pediatric blood culture bottles using an in-house-developed method. Organism identifications with a score of ≥1.6 were notified to treating clinicians. Therapeutic modifications that occurred after the communication of DIMT were reviewed through the electronic medical records. DIMT was performed on 530 pediatric positive blood culture bottles. Among 505 monomicrobial bottles, identifications from 298 (97.7%) deemed as bloodstream infections (BSI) and 189 (94.5%) as contaminations had DIMT notified to clinicians. All identifications were correct except for one Streptococcus mitis incorrectly reported as Streptococcus pneumoniae. Therapy modifications resulting from DIMT occurred in 27 (8.3%) patients with BSI. Deescalation from effective or ineffective broad-spectrum regimens occurred mainly in Enterococcus faecalis bacteremia, whereas appropriate escalation from an ineffective regimen with narrower spectrum occurred mainly in bacteremia caused by AmpC-ß-lactamase-producing Enterobacterales. Escalation therapy was instituted significantly faster than deescalation therapy (median time, 0.75 versus 10.5 h [P = 0.01]). DIMT also enabled clinicians to confirm contamination in nearly one-half of patients with contaminated blood cultures. Our DIMT method applied to positive pediatric blood culture bottles demonstrated reliable performance for the rapid identification of pathogens. Our DIMT approach allowed therapeutic optimization in BSI, especially involving microorganisms with intrinsic antibiotic resistance, and was helpful in the early identification of likely contaminants. IMPORTANCE We demonstrate the performance and early impact on the antimicrobial management of bloodstream infections of an inexpensive, in-house preparation method for direct identification of bloodstream pathogens in pediatric blood culture bottles by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry.

Fecal Carriage and Molecular Characterization of Carbapenemase-Producing Enterobacterales in the Pediatric Population in Qatar.

Whole-genome sequencing was used to characterize carbapenemase-producing Enterobacterales (CPE) strains recovered from rectal screening swab samples obtained from children at a tertiary-care pediatric hospital in Qatar during a 3-year period. A total of 72 CPE isolates recovered from 61 fecal carriers were characterized. Escherichia coli (47 isolates [65.3%]) and Klebsiella pneumoniae (22 isolates [30.6%]) were the most common species identified. High levels of genetic diversity were observed for both species. These 72 isolates produced 78 carbapenemases, characterized as either NDM-type (41 enzymes [52.6%]) or OXA-48-type (37 enzymes [47.4%]). NDM-5 (24 enzymes [30.8%]), NDM-1 (15 enzymes [19.2%]), and OXA-181 (15 enzymes [19.2%]) were the most common variants detected within each type. Twenty-three NDM producers exhibited difficult-to-treat resistance, compared with only 2 of the OXA-48 producers. Multiple comorbidities were identified in 88.5% of the patients, whereas recent travel history to countries in which CPE are endemic was documented for 57.4% of the patients. All 9 blaOXA-48-type-gene-containing E. coli sequence type 38 (ST38) strains were isolated from patients without international travel history. The mean quarterly incidence of fecal carriage decreased more than 6-fold after the implementation of coronavirus disease 2019 (COVID-19)-related international travel restrictions in Qatar in mid-March 2020. Our data suggest that NDM-type and OXA-48-type carbapenemases expressed by a large diversity of E. coli and K. pneumoniae genotypes are largely dominant in the pediatric population of Qatar. Although our data indicate successful local expansion of E. coli ST38 strains harboring blaOXA-244 genes, at least within health care settings, blaOXA-48-type and blaNDM-type genes appear to have been mainly introduced sporadically by asymptomatic carriers who visited or received health care in some nearby countries in which the genes are endemic. IMPORTANCE To the best of our knowledge, this is the first study addressing the molecular characteristics of CPE in a pediatric population in Qatar using whole-genome sequencing. Since several countries in the Arabian Peninsula share relatively similar demographic patterns and international links, it is plausible that the molecular characteristics of CPE in children, at least in the middle and eastern parts of the region, are similar to those observed in our study.

Nasopharyngeal Expression of Angiotensin-Converting Enzyme 2 and Transmembrane Serine Protease 2 in Children within SARS-CoV-2-Infected Family Clusters.

Lower levels of angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2) in the nasal epithelium of children may be related to a lower incidence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, compared to adults. However, no direct evidence is available to support this hypothesis. In this study, we compared the transcript levels of ACE2 and TMPRSS2 in nasopharyngeal swab samples (n = 234) from children and adult family members within SARS-CoV-2-exposed families and assessed the association with SARS-CoV-2 infection status. Transcript levels for ACE2, but not TMPRSS2, were higher in adults than in children (n = 129 adults and 105 children; P < 0.05). The expression of the two genes was not significantly different between SARS-CoV-2 positive and SARS-CoV-2 negative patients within the same age groups. However, in families with one or more SARS-CoV-2 positive adult family members, expression of both genes was significantly higher in SARS-CoV-2 positive children than in SARS-CoV-2 negative children (P < 0.05). By multivariate analysis, ACE2 expression adjusted for age and sex was significantly associated with SARS-CoV-2 infection in the overall population (odds ratio [OR], 1.112 [95% confidence interval [CI], 1.012 to 1.229]; P < 0.05). The degree of this association was higher (OR, 1.172 [95% CI, 1.034 to 1.347]; P < 0.05) in the subgroup of families with only SARS-CoV-2 positive adult family members. Our results suggest that children with lower levels of nasal ACE2 and TMPRSS2 are more likely to remain SARS-CoV-2 negative despite being exposed to a SARS-CoV-2 positive adult family member. IMPORTANCE ACE2 and TMPRSS2 are well established in the literature as SARS-CoV-2 entry factors. Recent data suggest that lower levels of nasal ACE2 in children may be associated with their lower incidence of coronavirus disease 2019 (COVID-19). In this study, using data from nasopharyngeal swab specimens from adult and pediatric members of families in which one or more members of the family had laboratory-confirmed SARS-CoV-2 infection, we show that children with lower levels of ACE2 and TMPRSS2 are more likely to remain SARS-CoV-2 negative despite being exposed to a SARS-CoV-2 positive adult family member. These results provide new insights into the roles of nasopharyngeal ACE2 and TMPRSS2 in acquiring SARS-CoV-2 infection, and they show that the differential expression of these genes in adults versus children may contribute to differential rates of SARS-CoV-2 infection in these populations.

Metabolomics in the Diagnosis and Prognosis of COVID-19.

Coronavirus disease 2019 (COVID-19) pandemic triggered an unprecedented global effort in developing rapid and inexpensive diagnostic and prognostic tools. Since the genome of SARS-CoV-2 was uncovered, detection of viral RNA by RT-qPCR has played the most significant role in preventing the spread of the virus through early detection and tracing of suspected COVID-19 cases and through screening of at-risk population. However, a large number of alternative test methods based on SARS-CoV-2 RNA or proteins or host factors associated with SARS-CoV-2 infection have been developed and evaluated. The application of metabolomics in infectious disease diagnostics is an evolving area of science that was boosted by the urgency of COVID-19 pandemic. Metabolomics approaches that rely on the analysis of volatile organic compounds exhaled by COVID-19 patients hold promise for applications in a large-scale screening of population in point-of-care (POC) setting. On the other hand, successful application of mass-spectrometry to detect specific spectral signatures associated with COVID-19 in nasopharyngeal swab specimens may significantly save the cost and turnaround time of COVID-19 testing in the diagnostic microbiology and virology laboratories. Active research is also ongoing on the discovery of potential metabolomics-based prognostic markers for the disease that can be applied to serum or plasma specimens. Several metabolic pathways related to amino acid, lipid and energy metabolism were found to be affected by severe disease with COVID-19. In particular, tryptophan metabolism via the kynurenine pathway were persistently dysregulated in several independent studies, suggesting the roles of several metabolites of this pathway such as tryptophan, kynurenine and 3-hydroxykynurenine as potential prognostic markers of the disease. However, standardization of the test methods and large-scale clinical validation are necessary before these tests can be applied in a clinical setting. With rapidly expanding data on the metabolic profiles of COVID-19 patients with varying degrees of severity, it is likely that metabolomics will play an important role in near future in predicting the outcome of the disease with a greater degree of certainty.

Evaluation of automated molecular tests for the detection of SARS-CoV-2 in pooled nasopharyngeal and saliva specimens.

BACKGROUND: Pooling of samples for SARS-CoV-2 testing in low-prevalence settings has been used as an effective strategy to expand testing capacity and mitigate challenges with the shortage of supplies. We evaluated two automated molecular test systems for the detection of SARS-CoV-2 RNA in pooled specimens. METHODS: Pooled nasopharyngeal and saliva specimens were tested by Qiagen QIAstat-Dx Respiratory SARS-CoV-2 Panel (QIAstat) or Cepheid Xpert Xpress SARS-CoV-2 (Xpert), and the results were compared to that of standard RT-qPCR tests without pooling. RESULTS: In nasopharyngeal specimens, the sensitivity/specificity of the pool testing approach, with 5 and 10 specimens per pool, were 77%/100% (n = 105) and 74.1%/100% (n = 260) by QIAstat, and 97.1%/100% (n = 250) and 100%/99.5% (n = 200) by Xpert, respectively. Pool testing of saliva (10 specimens per pool; n = 150) by Xpert resulted in 87.5% sensitivity and 99.3% specificity compared to individual tests. Pool size of 5 or 10 specimens did not significantly affect the difference of RT-qPCR cycle threshold (CT ) from standard testing. RT-qPCR CT values obtained with pool testing by both QIAstat and Xpert were positively correlated with that of individual testing (Pearson's correlation coefficient r = 0.85 to 0.99, p < 0.05). However, the CT values from Xpert were significantly stronger (p < 0.01, paired t test) than that of QIAstat in a subset of SARS-CoV-2 positive specimens, with mean differences of -4.3 ± 2.43 and -4.6 ± 2 for individual and pooled tests, respectively. CONCLUSION: Our results suggest that Xpert SARS-CoV-2 can be utilized for pooled sample testing for COVID-19 screening in low-prevalence settings providing significant cost savings and improving throughput without affecting test quality.

COVID-19 related multisystem inflammatory syndrome in children (MIS-C): a case series from a tertiary care pediatric hospital in Qatar.

BACKGROUND: Multisystem Inflammatory Syndrome in Children (MIS-C) is a severe complication of coronavirus disease 2019 (COVID-19) in children, which is increasingly being reported worldwide. Here we report the first case series of 7 children diagnosed with MIS-C in Qatar. METHODS: Clinical features and outcomes of COVID-19 positive patients admitted to Sidra Medicine, Qatar from June to October 2020, who met the WHO case definition for MIS-C were reviewed. RESULTS: The mean age in our case series was 5.6 years, of which 71.4% were males. All patients were previously healthy but had a history of COVID-19 infection. Fever, rash, vomiting and abdominal pain were the most common symptoms (70-100%). The average hospitalization was 12.9 days with no case fatalities. Laboratory findings included lymphopenia and thrombocytopenia in most patients, as well as evidence of coagulopathy and elevated inflammatory markers such as C-reactive protein, ferritin and procalcitonin. Many patients (71.4%) required inotropic support in intensive care, while only one required respiratory support. Although all patients had elevated cardiac biomarkers, cardiovascular involvement was observed in 42.9% of patients with one patient developing a giant coronary aneurysm. All patients received intravenous immunoglobulin (IVIG) and 86% of patients received corticosteroids, with two patients requiring treatment with IL-1 inhibitors. CONCLUSIONS: Our report is one of the first reports on MIS-C from Asia. Although clinical features and outcomes are not significantly different from those reported elsewhere, lack of case fatalities in our cohort may indicate that early recognition and prompt medical attention is necessary for a favorable outcome in MIS-C.

Peritoneal Dialysis-Associated Peritonitis Caused by Mycobacterium abscessus in Children-A Case Report.

Peritoneal dialysis (PD)-associated peritonitis constitutes a major complication associated with the procedure. PD-associated peritonitis caused by nontuberculous mycobacteria, usually as a result of an infection related to the PD catheter, has been reported in adults and is associated with significant complications and poor outcome. The management of PD-associated peritonitis caused by Mycobacterium abscessus is particularly challenging because this species is resistant to many antimicrobials commonly used to treat mycobacterial species. We present here the second reported case of PD-associated peritonitis caused by M. abscessus in children. Our patient was a 9-year-old boy with end-stage renal disease (ESRD) who presented with suspected peritonitis, and his PD fluid cultures eventually grew M. abscessus. The patient received a 3-week course of triple therapy with clarithromycin, amikacin, and meropenem in addition to PD catheter removal. The infection completely resolved even though a susceptibility report at the end of treatment revealed that the isolate was resistant to clarithromycin and had decreased susceptibility to carbapenems. Our observations suggest that PD catheter removal is important in PD-associated peritonitis caused by M. abscessus in children and that more studies are needed to define the optimal length of treatment.

Virome-wide serological profiling reveals association of herpesviruses with obesity.

The relationship between viral infection and obesity has been known for several decades but epidemiological data is limited to only a few viral pathogens. The association between obesity and a wide range of viruses was assessed using VirScan, a pan-viral serological profiling tool. Serum specimens from 457 Qatari adults (lean = 184; obese = 273) and 231 Qatari children (lean = 111; obese = 120) were analyzed by VirScan. Associations with obesity were determined by odds ratio (OR) and Fisher's test (p values), and by multivariate regression analysis to adjust for age and gender. Although there was no association of viral infections with obesity in the pediatric population, a nominal association of obesity with seropositivity to members of the Herpesviridae family is observed for the adult population (OR = 1.5-3.3; p < 0.05). After adjusting p values for multiple comparisons (Bonferroni correction) the odds of being obese is significantly higher in herpes simplex virus 1 (HSV-1) seropositive Qatari adults (OR = 3.3; 95% CI 2.15-4.99; p = 2.787E - 08). By VirScan, the sero-prevalence of HSV1 is 81.3% and 57.1% among Qatari obese and lean adult populations, respectively. Higher prevalence of antibodies against several peptide epitopes of HSV-1/2 is positively associated with obesity (OR = 2.35-3.82; p ≤ 3.981E - 05). By multivariate regression analysis, HSV-1 was independently associated with obesity irrespective of age and gender. Our results suggest that obesity among Qataris may be associated with a higher prevalence of herpesvirus infections, in particular HSV-1. Furthermore, the high prevalence of antibodies against peptide antigens specific to HSV-1 and -2 in the obese population suggests that these viral peptides may play a role in adipogenesis. Further studies with these candidate peptides in cell culture or animal models may confirm their adipogenic roles.

Molecular Characterization of Extended-Spectrum ß-Lactamase-Producing Escherichia coli and Klebsiella pneumoniae Among the Pediatric Population in Qatar.

INTRODUCTION: Although extended-spectrum ß-lactamase (ESBL)-producing Enterobacterales are a public health problem in the Arabian Peninsula, data on the molecular characteristic of their antimicrobial resistance determinants in children is limited. AIM: To determine the molecular characteristics of ESBL-producing Escherichia coli and Klebsiella pneumoniae in the pediatric population of Qatar. METHODS: Whole-genome sequencing was performed on ESBL-producing E. coli and K. pneumoniae isolates recovered from screening and clinical specimens from pediatric patients at Sidra Medicine in Doha from January to December 2018. RESULTS: A total of 327 ESBL producers were sequenced: 254 E. coli and 73 K. pneumoniae. Non-susceptibility rates to non-ß-lactam antibiotics for both species were 18.1 and 30.1% for gentamicin, 0.8 and 4.1% for amikacin, 41.3 and 41.1% for ciprofloxacin, and 65.8 and 76.1% for cotrimoxazole. The most common sequence types (STs) were ST131 (16.9%), ST38 and ST10 (8.2% each) in E. coli and ST307 (9.7%), and ST45 and ST268 (6.9% each) in K. pneumoniae. CTX-M type ESBLs were found in all but one isolate, with CTX-M-15 accounting for 87.8%. Among other ß-lactamases, TEM-1B and OXA-1 were coproduced in 41 and 19.6% of isolates. The most common plasmid-mediated quinolone resistance genes cocarried were qnr A/B/E/S (45.3%). Ninety percent of gentamicin non-susceptible isolates harbored genes encoding AAC(3) enzymes, mainly aac(3)-IIa. Only two of 57 isolates harboring aac(6')-Ib-cr were non-susceptible to amikacin. Chromosomal mutations in genes encoding DNA gyrase and topoisomerase IV enzymes were detected in 96.2% fluoroquinolone-non-susceptible E. coli and 26.7% fluoroquinolone-non-susceptible K. pneumoniae. CONCLUSION: Our data show that CTX-M enzymes are largely the most prevalent ESBLs in children in Qatar with a predominance of CTX-M-15. Carbapenem-sparing options to treat ESBL infections are limited, given the frequent coproduction of OXA-1 and TEM-1B enzymes and coresistance to antibiotic classes other than ß-lactams.