Evaluation of optimal urine screening and confirmation cut-off values for opiates, at a national reference laboratory

Objectives To obtain optimal immunoassay screening and LC-MS/MS confirmation cut-offs for opiate group tests to reduce false positive (FP) and false negative (FN) rates. Methods A total of 126 urine samples, −50 opiate screening negative, 76 positive according to the threshold of 300 ng/mL by CEDIA method – were confirmed by a full-validated in-house LC-MS/MS method. Sensitivity, specificity, FP, and FN rates were determined at cut-off concentrations of both 300 and 2,000 ng/mL for morphine and codeine, and 10 ng/mL for heroin metabolite 6-mono-acetyl-morphine (6-MAM). Results All CEDIA opiate negative urine samples were negative for morphine, codeine and 6-MAM. Although sensitivity was 100% for each cut-off; specificity was 54.9% at CEDIA cut-off 300 ng/mL vs. LC-MS/MS cut-off 300 ng/mL and, 75% at CEDIA cut-off 2,000 ng/mL vs. LC-MS/MS cut-off 2,000 ng/mL. False positive rate was highest (45.1%) at CEDIA cut-off 300 ng/mL. At CEDIA cut-off 2,000 ng/mL vs. LC-MS/MS cut-off 300 ng/mL, specificity increased to 82.4% and FP rate decreased to 17.6%. All 6-MAM positive samples had CEDIA concentration ≥2,000 ng/mL. Conclusions 2,000 ng/mL for screening and 300 ng/mL for confirmation cut-offs are the most efficient thresholds for the lowest rate of FP opiate results.

Impact of the COVID-19 pandemic on tuberculosis national reference laboratory services in the WHO European Region, March to November 2020

We assessed the impact of COVID-19 on diagnostic services for tuberculosis (TB) by national reference laboratories in the WHO European Region. Of 35 laboratories, 30 reported declines in TB sample numbers, amounting up to > 50% of the pre-COVID-19 volumes. Sixteen reported reagent or consumable shortages. Nineteen reallocated ressources to SARS-CoV-2 testing, resulting in an overall increase in workload, largely without a concomitant increase in personnel (n = 14). This poses a risk to meeting the 2025 milestones of the End TB Strategy.

Detection of markers predictive of macrolide and fluoroquinolone resistance in Mycoplasma genitalium from patients attending sexual health services

ObjectivesThis study sought to provide data on the prevalence of macrolide (23S rRNA) and fluoroquinolone (parC) resistance-associated mutations seen in Mycoplasma genitalium-positive specimens received in the UK national reference laboratory.MethodsIn total, 2580 clinical specimens from patients with suspected or confirmed M. genitalium infection were received at the national reference laboratory between September 2017 and November 2018. M. genitalium-positive clinical specimens were identified using a reverse transcription-PCR targeting two M. genitalium genes: MgPa and gap. Resistance-associated single nucleotide poylmorphisms were sought in all positive specimens by sequence analysis of the 23S rRNA and parC genes.ResultsEighteen per cent (458 of 2580) of clinical specimens were positive for M. genitalium and 389 had sequence data for both macrolide and fluoroquinolone resistance markers. Of these, 71% (275 of 389) had macrolide resistance-associated mutations, 8% (31 of 389) had fluoroquinolone resistance-associated mutations (S83I/R and D87Y/N) and 7% (26 of 389) had mutations associated with resistance to both antimicrobials. Only 28% (108 of 389) had no mutations associated with resistance to either class of antibiotic. Five specimens had mutations of unknown clinical significance in the parC gene (eg, G81C and S83N).ConclusionsMutations associated with resistance to macrolides were very frequent. By contrast, susceptibility to the second-line treatment, moxifloxacin (a fluoroquinolone), was estimated at 92% based on the absence of resistance-associated mutations. The few specimens with mutations of unknown clinical significance in the parC gene were excluded from the analysis and so the actual level of fluoroquinolone susceptibility may be slightly lower than that reported here. Surveillance of antimicrobial resistance in M. genitalium is imperative for this to remain a treatable infection.

Implementation of WHO Global Antimicrobial Resistance Surveillance System in Uganda: National Surveillance Report, 2015 to 2020 (Preprint)

BACKGROUND Antimicrobial resistance is an emerging public health crisis in Uganda. The WHO Global Action Plan recommends that countries develop and implement National Actions Plans for AMR. We describe the establishment of the national AMR program in Uganda and present earlier sensitivity results from the program. OBJECTIVE The objective of the national surveillance programme is the systematic, continuous collection, analysis and interpretation of antimicrobial resistance data. METHODS A systematic qualitative description of the process and progress made in the establishment of the national AMR program is provided, detailing progress made since 2015 to 2020. This is followed with reporting of the findings of the isolates that are collected from the sentinel AMR surveillance sites. The identification and AST of bacterial isolates presented was done using standard methods at both the sentinel sites and the national reference laboratory. RESULTS Progress has been made in establishment of the national AMR program and implementation of the GLASS protocol is ongoing. A national coordinating centre and focal person have been established, a national reference laboratory has been designated, WHO net set up, sentinel AMR surveillance sites have been established with both data and laboratory quality assurance incorporated. Uganda has progressively submitted data to the GLASS reporting system. 19,216 isolates from WHO GLASS priority specimens were collected of which 22.95% (n=4,411) were community acquired infections (CAIs), 9.5% (n=1,818) had hospital acquired infections (HAIs) with 68.57% (n=12,987) being of unknown origin. The highest proportion of the specimens was blood (n=12,398, 64.5%) followed by urine (n=5,278, 27.5%), and then by stool (n=1,266, 6.6%), while, the least proportion were uro-genital swabs (n=274, 1.4%). The mean age was 19.1 (SD=19.8) years while the median was 13 (IQR: 28). Approximately 49.1% of the participants were female and 50.5% were male. Participants with CAIs were older than those with HAIs i.e. Mean: 28.0 (SD=18.6), Median: 26, IQR: 20.5 vs. Mean: 17.3 (SD=20.9) Median 8 IQR: 26. All gram-negative (E. coli, K. pneumoniae, N. gonorhoeae) and gram-positive (S. aureus, Enterococcus sp.) bacteria with AST done showed resistance to each of the tested antibiotics. CONCLUSIONS We demonstrate that systematic capacity building for implementation of the WHO GLASS protocol is feasible in a low resource setting. CLINICALTRIAL NA

The COVID-19 Epidemic in Madagascar: clinical description and laboratory results of the first wave, March-September 2020

Background: Following the first detection of SARS-CoV-2 in passengers arriving from Europe on 19 March 2020, Madagascar took several mitigation measures to limit the spread of the virus in the country. Methods: Nasopharyngeal and/or oropharyngeal swabs were collected from travellers to Madagascar, suspected SARS-CoV-2 cases, and contact of confirmed cases. Swabs were tested at the national reference laboratory using real-time RT-PVR. Data collected from patients were entered in an electronic database for subsequent statistical analysis. All distribution of laboratory confirmed cases were mapped and six genomes of viruses were fully sequenced. Results: Overall, 26,415 individuals were tested for SARS-CoV-2 between 18 March and 18 September 2020, of whom 21.0% (5,553/26,145) returned positive. Among laboratory-confirmed SARS-CoV-2 positive patients, the median age was 39 years (CI95%: 28-52), and 56.6% (3,311/5,553) were asymptomatic at the time of sampling. The probability of testing positive increased with age with the highest adjusted odds ratio of 2.2 [95% CI: 1.9-2.5] for individuals aged 49 years and more. Viral strains sequenced belong to clades 19A, 20A, and 20B in favour of several independent introduction of viruses. Conclusions. Our study describes the first wave of the COVID-19 in Madagascar. Despite early strategies in place Madagascar could not avoid the introduction and spread of the virus. More studies are needed to estimate the true burden of disease and make public health recommendations for a better preparation to another wave.

Retrospective analysis on confirmation rates for referred positive rotavirus samples in England, 2016 to 2017: implications for diagnosis and surveillance

Background Rapid diagnostic tests are commonly used by hospital laboratories in England to detect rotavirus (RV), and results are used to inform clinical management and support national surveillance of the infant rotavirus immunisation programme since 2013. In 2017, the Public Health England (PHE) national reference laboratory for enteric viruses observed that the presence of RV could not be confirmed by PCR in a proportion of RV-positive samples referred for confirmatory detection. Aim We aimed to compare the positivity rate of detection methods used by hospital laboratories with the PHE confirmatory test rate. Methods Rotavirus specimens testing positive at local hospital laboratories were re-tested at the PHE national reference laboratory using a PCR test. Confirmatory results were compared to original results from the PHE laboratory information management system. Results Hospital laboratories screened 70.1% (2,608/3,721) of RV samples using immunochromatographic assay (IC) or rapid tests, 15.5% (578/3,721) using enzyme immunoassays (EIA) and 14.4% (535/3,721) using PCR. Overall, 1,011/3,721 (27.2%) locally RV-positive samples referred to PHE in 2016 and 2017 failed RV detection using the PHE reference laboratory PCR test. Confirmation rates were 66.9% (1,746/2,608) for the IC tests, 87.4% (505/578) for the EIA and 86.4% (465/535) for the PCR assays. Seasonal confirmation rate discrepancies were also evident for IC tests. Conclusions This report highlights high false positive rates with the most commonly used RV screening tests and emphasises the importance of implementing verified confirmatory tests for RV detections. This has implications for clinical diagnosis and national surveillance.

A Ten-Year Review of Carbapenemase Producing Enterobacterales (CPE) in London, United Kingdom

Background: To determine the pattern of CPE observed in a single region in the United Kingdom. Methods: From 2009 to 2018, clinical laboratories in England were requested to send suspected CPE from all sites to the national reference laboratory for confirmation and investigation of carbapenem resistance mechanism(s). Isolates of Enterobacterales from London laboratories and confirmed to have 1 or more carbapenemase genes were included in the analysis. Result: Between 2009 and 2018, 5,133 isolates were confirmed to produce a carbapenemase; at least 1 CPE was identified in every London Laboratory and hospital. Confirmations increased from 28 isolates in 2009 to 1857 in 2018 and with a sharp rise after the introduction of the ‘PHE toolkit’ in 2013 (Fig. 1). Most CPE (2655, 51.7%) were from rectal screens (the 3 most frequently identified carbapenemase families were OXA-48–like in 1,263 isolates, NDM in 971 and IMP in 128), 631 (12.3%) were from urine samples, 180 (3.5%) from blood cultures, 103 (2.0%) from sputum specimens and the remainder (1564, 30.5%) were swabs, fluids and tissues from various body sites. Moreover, 51 CPE (1%) were identified from environmental swabs. Isolates were predominantly Klebsiella spp (2,525, 49%; 2,088 were K. pneumoniae), followed by Escherichia coli (1,434, 27.9%), Enterobacter spp (746, 14.5%; 605 were E. cloacae complex), and Citrobacter spp (349, 6.8%); 10 other species contributed smaller numbers. Within the carbapenemase families, OXA-48-like enzymes predominated overall (2303, 44.9%), followed by NDM (1822, 35.5%), IMP (313, 6.1%), VIM (207, 4.0%), NDM+OXA-48-like (205, 4.0%), and KPC (196, 3.8%). The first detection of a CPE with 2 distinct enzymes occurred in 2012 (OXA-48-like and NDM) and since then 235 co-detections have been identified; 233 related to OXA-48-like with another gene. Conclusion: The first CPE isolate in London was identified in 2003, a Klebsiella spp with a VIM enzyme. The number of isolates submitted to the national reference laboratory has continued to increase year on year. VIM and NDM carbapenemases predominated in the early years, because of their association with several outbreaks; these have now been overwhelmed by OXA-48-like detections and outbreaks. The increasing numbers of CPE with a combination of a metallo- and a non-metallo carbapenemase increases the therapeutic challenges to treat infected patients. Bacteremia caused by CPE remains rare, suggesting that infection prevention and control efforts are having some impact. However, as colonization prevalence increases, the number of clinical infections will rise in the future unless control measures to limit transmission and spread are improved.Funding: NoneDisclosures: None