Direct Blood PCR in Combination with Nucleic Acid Lateral Flow Immunoassay for Detection of Plasmodium Species in Settings Where Malaria Is Endemic

Declining malaria transmission and known difficulties with current diagnostic tools for malaria, such as microscopy and rapid diagnostic tests (RDTs) in particular at low parasite densities, still warrant the search for sensitive diagnostic tests. Molecular tests need substantial simplification before implementation in clinical settings in countries where malaria is endemic. Direct blood PCR (db-PCR), circumventing DNA extraction, to detect Plasmodium was developed and adapted to be visualized by nucleic acid lateral flow immunoassay (NALFIA). The assay was evaluated in the laboratory against samples from confirmed Sudanese patients (n = 51), returning travelers (n = 214), samples from the Dutch Blood Bank (n = 100), and in the field in Burkina Faso (n = 283) and Thailand (n = 381) on suspected malaria cases and compared to RDT and microscopy. The sensitivity and specificity of db-PCR-NALFIA compared to the initial diagnosis in the laboratory were 94.4% (95% confidence interval [CI] = 0.909 to 0.969) and 97.4% (95% CI = 0.909 to 0.969), respectively. In Burkina Faso, the sensitivity was 94.8% (95% CI = 0.88.7 to 97.9%), and the specificity was 82.4% (95% CI = 75.4 to 87.7%) compared to microscopy and 93.3% (95% CI = 87.4 to 96.7%) and 91.4% (95% CI = 85.2 to 95.3%) compared to RDT. In Thailand, the sensitivity and specificity were 93.4% (CI = 86.4 to 97.1%) and 90.9 (95% CI = 86.7 to 93.9%), respectively, compared to microscopy and 95.6% (95% CI = 88.5 to 98.6%) and 87.1% (95% CI = 82.5 to 90.6) compared to RDT. db-PCR-NALFIA is highly sensitive and specific for easy and rapid detection of Plasmodium parasites and can be easily used in countries where malaria is endemic. The inability of the device to discriminate Plasmodium species requires further investigation.     

Diagnostic accuracy of a rapid E1-antigen test for chikungunya virus infection in a reference setting

ObjectivesRapid diagnostic tests targeting virus-specific antigen could significantly enhance the diagnostic capacity for chikungunya virus infections. We evaluated the accuracy of an immunochromatographic antigen test for diagnosis of chikungunya in a reference laboratory for arboviruses.MethodsAn immunochromatographic rapid test that uses mouse monoclonal antibodies as a tracer against the E1-envelope protein of chikungunya (ARKRAY, Inc. Kyoto, Japan) was evaluated. Sensitivity was tested in sera from travellers with RT-PCR confirmed chikungunya virus infection (Eastern/Central/Southern African (ECSA) genotype) (n=9) and from patients diagnosed during the 2014-2015 chikungunya outbreak on Aruba (Asian genotype, n=30). Samples from patients with other febrile and non-febrile illnesses (n=26), sera spiked with Flavivirus and Alphavirus reference strains (n=13, including non-spiked serum), and samples containing other selected pathogens (n=20) were used to test specificity of the E1-antigen test.ResultsSensitivity of the E1-antigen test was 8/9 (88.9%, 95% CI 56.5–98.0) for the ECSA genotype, but only 10/30 (33.3%, 95% CI 19.2-51.2) for the Asian genotype. Overall diagnostic specificity was 49/59 (83.1%, 95% CI 71.5-90.5).ConclusionsThe E1-antigen test we evaluated had fair diagnostic sensitivity for ECSA genotype chikungunya, but low sensitivity for Asian genotype, and poor overall specificity. Antibodies that react across genotypes will be required for further development of a rapid test for chikungunya. Performance of new tests should be evaluated against different chikungunya genotypes.     

Comparison of five methods for detection of carbapenemases in Enterobacterales with proposal of a new algorithm

ObjectivesThe aim of this study was to evaluate the performance of five different carbapenemase tests and to develop an algorithm which will permit the detection of most common and rare carbapenemases in routine microbiology laboratories.MethodsThe immunochromatographic tests CARBA-5 (NG), RESIST-4 O.K.N.V. (Coris), the colorimetric β-CARBA (BioRad), a newly developed carbapenem-inactivation method (CIM) supplemented with zinc (zCIM), and the Xpert Carba-R (Cepheid) were challenged with a collection of 189 molecularly characterized Enterobacterales isolates, including 146 carbapenemase producers (CPE): VIM (n = 48), OXA-48-like (n = 40), NDM (n = 29), KPC (n = 13), IMI (n = 9), IMP (n = 9), OXA-58 (n = 2), and GES (n = 2).ResultsThe overall sensitivity/specificity values for the five carbapenemase detection tests were 84.2% (CI 77.6–89.2%)/100% (CI 91.8–100%) for RESIST-4, 88.2% (CI 82.1–92.4%)/100% (CI 91.8–100%) for CARBA-5, 88.2% (CI 82.1–92.4%)/100% (CI 91.8–100%) for Xpert Carba-R, 73.7% (CI 66.2–80.0%)/100% (CI 93.4–99.0%) for β-CARBA, and 97.4% (CI 87.9–99.6%)/97.7% (CI 87.9–99.6%) for zCIM. The four common carbapenemases (KPC, OXA-48-like, NDM, and VIM) were detected with ≥97.6% sensitivity by all tests except for β-CARBA (76.6% (CI 68.4–83.2%)). IMI and GES were only detected by zCIM (sensitivity 90.9% (CI 62.3–98.4%)). Based on these results a new algorithm was developed, consisting of an immunochromatographic assay as the first test followed by zCIM, which allows detection of 99.3% of all carbapenemases assessed.ConclusionsExcept for β-CARBA, all methods showed excellent sensitivity/specificity for the detection of the four most frequent carbapenemases. With the new algorithm, rare variants can also be detected. It is rapid, simple, and inexpensive and can be performed in any microbiology laboratory, as no PCR equipment is required.     

Loop-Mediated Isothermal Amplification for Rickettsia typhi (the Causal Agent of Murine Typhus): Problems with Diagnosis at the Limit of Detection

Murine typhus is a flea-borne disease of worldwide distribution caused by Rickettsia typhi. Although treatment with tetracycline antibiotics is effective, treatment is often misguided or delayed due to diagnostic difficulties. As the gold standard immunofluorescence assay is imperfect, we aimed to develop and evaluate a loop-mediated isothermal amplification (LAMP) assay. LAMP assays have the potential to fulfill the WHO ASSURED criteria (affordable, sensitive, specific, user friendly, robust and rapid, equipment free, deliverable to those who need them) for diagnostic methodologies, as they can detect pathogen-derived nucleic acid with low technical expenditure. The LAMP assay was developed using samples of bacterial isolates (n = 41), buffy coat specimens from R. typhi PCR-positive Lao patients (n = 42), and diverse negative controls (n = 47). The method was then evaluated prospectively using consecutive patients with suspected scrub typhus or murine typhus (n = 266). The limit of detection was ∼40 DNA copies/LAMP reaction, with an analytical sensitivity of <10 DNA copies/reaction based on isolate dilutions. Despite these low cutoffs, the clinical sensitivity was disappointing, with 48% (95% confidence interval [95% CI], 32.5 to 62.7%) (specificity, 100% [95% CI, 100 to 100%]) in the developmental phase and 33% (95% CI, 9.2 to 56.8%) (specificity, 98.5% [95% CI, 97.0% to 100%]) in the prospective study. This low diagnostic accuracy was attributed to low patient R. typhi bacterial loads (median, 210 DNA copies/ml blood; interquartile range, 130 to 500). PCR-positive but LAMP-negative samples demonstrated significantly lower bacterial loads than LAMP-positive samples. Our findings highlight the diagnostic challenges for diseases with low pathogen burdens and emphasize the need to integrate pathogen biology with improved template production for assay development strategies.     

Incidence and clinical variables associated with streptococcal throat infections: a prospective diagnostic cohort study

Background

Management of pharyngitis is commonly based on features which are thought to be associated with Lancefield group A beta-haemolytic streptococci (GABHS) but it is debatable which features best predict GABHS. Non-group A strains share major virulence factors with group A, but it is unclear how commonly they present and whether their presentation differs.

Aim

To assess the incidence and clinical variables associated with streptococcal infections.

Design and setting

Prospective diagnostic cohort study in UK primary care.

Method

The presence of pathogenic streptococci from throat swabs was assessed among patients aged ≥5 years presenting with acute sore throat.

Results

Pathogenic streptococci were found in 204/597 patients (34%, 95% CI = 31 to 38%): 33% (68/204) were non-group A streptococci, mostly C (n = 29), G (n = 18) and B (n = 17); rarely D (n = 3) and Streptococcus pneumoniae (n = 1). Patients presented with similar features whether the streptococci were group A or non-group A. The features best predicting A, C or G beta-haemolytic streptococci were patient’s assessment of severity (odds ratio [OR] for a bad sore throat 3.31, 95% CI = 1.24 to 8.83); doctors’ assessment of severity (severely inflamed tonsils OR 2.28, 95% CI = 1.39 to 3.74); absence of a bad cough (OR 2.73, 95% CI = 1.56 to 4.76), absence of a coryza (OR 1.54, 95% CI = 0.99 to 2.41); and moderately bad or worse muscle aches (OR 2.20, 95% CI = 1.41 to 3.42).

Conclusion

Non-group A strains commonly cause streptococcal sore throats, and present with similar symptomatic clinical features to group A streptococci. The best features to predict streptococcal sore throat presenting in primary care deserve revisiting.     

Insomnia with objective short sleep duration is associated with cognitive impairment: a first look at cardiometabolic contributors to brain health

Study ObjectivesInsomnia with objective short sleep duration has been previously associated with adverse cardiometabolic health outcomes as well as poorer cognitive performance in otherwise noncognitively impaired adults. However, studies demonstrating an increased prevalence of cognitive impairment (CI) in this insomnia phenotype are lacking.MethodsWe analyzed data from Penn State Adult Cohort (N = 1,524; 48.9 ± 13.4 years; 53.4% women). Self-reported sleep difficulty was defined as normal sleep (n = 899), poor sleep (n = 453), and chronic insomnia (n = 172). Objective short sleep duration was defined as less than 6-h of sleep, based on in-lab, 8-h polysomnography. CI (n = 155) and possible vascular cognitive impairment (pVCI, n = 122) were ascertained using a comprehensive neuropsychological battery. Analyses adjusted for age, sex, race, education, body mass index, apnea/hypopnea index, smoking, alcohol, psychoactive medication, and mental and physical health problems.ResultsParticipants who reported poor sleep or chronic insomnia and slept objectively less than 6 hours were associated with a 2-fold increased odds of CI (OR = 2.06, 95% confidence limits [CL] = 1.15–3.66 and OR = 2.18, 95% CL = 1.07–4.47, respectively) and of pVCI (OR = 1.94, 95% CL = 1.01–3.75 and OR = 2.33, 95% CL = 1.07–5.06, respectively). Participants who reported poor sleep or chronic insomnia and slept objectively more than 6 hours were not associated with increased odds of either CI (OR = 0.72, 95% CL = 0.30–1.76 and OR = 0.75, 95% CL = 0.21–2.71, respectively) or pVCI (OR = 1.08, 95% CL = 0.42–2.74 and OR = 0.76, 95% CL = 0.16–3.57, respectively).ConclusionsInsomnia with objective short sleep duration is associated with an increased prevalence of CI, particularly as it relates to cardiometabolic health (i.e. pVCI). These data further support that this insomnia phenotype may be a more biologically severe form of the disorder associated with cardiovascular, cerebrovascular, and neurocognitive morbidity.     

Improved COVID-19 ICU admission and mortality outcomes following treatment with statins: a systematic review and meta-analysis

IntroductionApproximately 1% of the world population has now been infected by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus disease 2019 (COVID-19). With cases still rising and vaccines just beginning to rollout, we are still several months away from seeing reductions in daily case numbers, hospitalisations, and mortality. Therefore, there is a still an urgent need to control the disease spread by repurposing existing therapeutics. Owing to antiviral, anti-inflammatory, immunomodulatory, and cardioprotective actions, statin therapy has been considered as a plausible approach to improve COVID-19 outcomes.Material and methodsWe carried out a meta-analysis to investigate the effect of statins on 3 COVID-19 outcomes: intensive care unit (ICU) admission, tracheal intubation, and death. We systematically searched the PubMed, Web of Science, Scopus, and ProQuest databases using keywords related to our aims up to November 2, 2020. All published observational studies and randomised clinical trials on COVID-19 and statins were retrieved. Statistical analysis with random effects modelling was performed using STATA16 software.ResultsThe final selected studies (n = 24 studies; 32,715 patients) showed significant reductions in ICU admission (OR = 0.78, 95% CI: 0.58–1.06; n = 10; I² = 58.5%) and death (OR = 0.70, 95% CI: 0.55–0.88; n = 21; I² = 82.5%) outcomes, with no significant effect on tracheal intubation (OR = 0.79; 95% CI: 0.57–1.11; n = 7; I²= 89.0%). Furthermore, subgroup analysis suggested that death was reduced further by in-hospital application of stains (OR = 0.40, 95% CI: 0.22–0.73, n = 3; I² = 82.5%), compared with pre-hospital use (OR = 0.77, 95% CI: 0.60–0.98, n = 18; I² = 81.8%).ConclusionsThese findings call attention to the need for systematic clinical studies to assess both pre- and in-hospital use of statins as a potential means of reducing COVID-19 disease severity, particularly in terms of reduction of ICU admission and total mortality reduction.     

Risk Factors for Campylobacteriosis in Two Washington State Counties with High Numbers of Dairy Farms

Campylobacteriosis is a frequently reported, food-borne, human bacterial disease that can be associated with ruminant reservoirs, although public health messages primarily focus on poultry. In Washington State, the two counties with the highest concentrations of dairy cattle also report the highest incidences of campylobacteriosis. Conditional logistic regression analysis of case-control data from both counties found living or working on a dairy farm (odds ratio [OR], 6.7 [95% confidence interval [CI], 1.7 to 26.4]) and Hispanic ethnicity (OR, 6.4 [95% CI, 3.1 to 13.1]) to have the strongest significant positive associations with campylobacteriosis. When the analysis was restricted to residents of one county, Hispanic ethnicity (OR, 9.3 [95% CI, 3.9 to 22.2]), contact with cattle (OR, 5.0 [95% CI, 1.3 to 19.5]), and pet ownership (OR, 2.6 [95% CI, 1.1 to 6.3]) were found to be independent risk factors for disease. Campylobacter jejuni isolates from human (n = 65), bovine (n = 28), and retail poultry (n = 27) sources from the same counties were compared using multilocus sequence typing. These results indicated that sequence types commonly found in human isolates were also commonly found in bovine isolates. These findings suggest that, in areas with high concentrations of dairy cattle, exposure to dairy cattle may be more important than food-borne exposure to poultry products as a risk for campylobacteriosis.     

Simple Objective Detection of Human Lyme Disease Infection Using Immuno-PCR and a Single Recombinant Hybrid Antigen

A serology-based tiered approach has, to date, provided the most effective means of laboratory confirmation of clinically suspected cases of Lyme disease, but it lacks sensitivity in the early stages of disease and is often dependent on subjectively scored immunoblots. We recently demonstrated the use of immuno-PCR (iPCR) for detecting Borrelia burgdorferi antibodies in patient serum samples that were positive for Lyme disease. To better understand the performance of the Lyme disease iPCR assay, the repeatability and variability of the background of the assay across samples from a healthy population (n = 36) were analyzed. Both of these parameters were found to have coefficients of variation of <3%. Using eight antigen-specific iPCR assays and positive call thresholds established for each assay, iPCR IgM and/or IgG diagnosis from Lyme disease patient serum samples (n = 12) demonstrated a strong correlation with that of 2-tier testing. Furthermore, a simplified iPCR approach using a single hybrid antigen and detecting only IgG antibodies confirmed the 2-tier diagnosis in the Lyme disease patient serum samples (n = 12). Validation of the hybrid antigen IgG iPCR assay using a blinded panel of Lyme disease and non-Lyme disease patient serum samples (n = 92) resulted in a sensitivity of 69% (95% confidence interval [CI], 50% to 84%), compared to that of the 2-tier analysis at 59% (95% CI, 41% to 76%), and a specificity of 98% (95% CI, 91% to 100%) compared to that of the 2-tier analysis at 97% (95% CI, 88% to 100%). A single-tier hybrid antigen iPCR assay has the potential to be an improved method for detecting host-generated antibodies against B. burgdorferi.     

Helicobacter pylori Infection in Infants and Toddlers in South America: Concordance between [13C]Urea Breath Test and Monoclonal H. pylori Stool Antigen Test

Accurate noninvasive tests for diagnosing Helicobacter pylori infection in very young children are strongly required. We investigated the agreement between the [¹³C]urea breath test ([¹³C]UBT) and a monoclonal ELISA (HpSA) for detection of H. pylori antigen in stool. From October 2007 to July 2011, we enrolled 414 infants (123 from Brazil and 291 from Peru) of ages 6 to 30 months. Breath and stool samples were obtained at intervals of at least 3 months from Brazilian (n = 415) and Peruvian (n = 908) infants. [¹³C]UBT and stool test results concurred with each other in 1,255 (94.86%) cases (kappa coefficient = 0.90; 95% confidence interval [CI] = 0.87 to 0.92). In the H. pylori-positive group, delta-over-baseline (DOB) and optical density (OD) values were positively correlated (r = 0.62; P < 0.001). The positivity of the tests was higher (P < 0.001; odds ratio [OR] = 6.01; 95% CI = 4.50 to 8.04) in Peru (546/878; 62.2%) than in Brazil (81/377; 21.5%) and increased with increasing age in Brazil (P = 0.02), whereas in Peru it decreased with increasing age (P < 0.001). The disagreement between the test results was associated with birth in Brazil and female gender but not with age and diarrhea. Our results suggest that both [¹³C]UBT and the stool monoclonal test are reliable for diagnosing H. pylori infection in very young children, which will facilitate robust epidemiological studies in infants and toddlers.     

Evaluation of recombinant leptospiral surface antigen (Lsa27) lipoprotein for serodiagnosis of human leptospirosis by latex agglutination test

PurposeLeptospirosis has wide clinical presentations often mimicking other illnesses, thus rapid and simple diagnostics will have facilitated the initial patient management and therapy compared to other inaccessible and laborious tests/assays.MethodIn this study, the sensitized latex beads coated with purified recombinant outer membrane (OM)-leptospiral surface antigen (Lsa27) lipoprotein of pathogenic Leptospira was evaluated as a diagnostic antigen in latex agglutination test (LAT) for the detection of anti-leptospiral antibodies in the human sera. The prepared rLsa27 latex beads were evaluated with the confirmed microscopic agglutination test (MAT) reactive (at 1:50) Leptospira-specific positive (n = 42) and non-reactive negative (n = 80) sera from human cases suspected of leptospirosis with the history of pyrexia of unknown origin.ResultThe results revealed the relative diagnostic sensitivity of 90.48 % (confidence interval (CI) at 95 % : 77.4–97.3 %) and diagnostic specificity of 91.35 % (CI at 95 %: 82.8–96.4 %), with an accuracy of 90.98 % (CI at 95 %: 84.44–95.41 %), and the kappa value of 0.8036 ± 0.056 SE (CI at 95 %: 0.69–0.91) with a substantial agreement against gold standard serological MAT.ConclusionThe findings suggest that the rLsa27 protein-based LAT can be useful as a simple rapid screening diagnostic test for the detection of anti-leptospiral antibodies in the sera of humans. This rapid test can be complemented by other confirmatory diagnostics for the early detection of Leptospira antibodies which may in turn help in the prompt treatment and mitigates the public health problem at primary health care level.     

Pediatric Human Immunodeficiency Virus Screening in an African District Hospital

In order to evaluate alternative tests and strategies to simplify pediatric human immunodeficiency virus (HIV) screening at the district hospital level, a cross-sectional exploratory study was organized in the Democratic Republic of the Congo. Venous and capillary phlebotomies were performed on 941 Congolese children, aged 1 month to 12 years (153 children under 18 months and 788 children more than 18 months old). The HIV prevalence rate was 4.7%. An algorithm for children more than 18 months old, using serial rapid tests (Determine, InstantScreen, and Uni-Gold) performed on capillary blood stored in EDTA tubes, had a sensitivity of 100.0% (95% confidence interval [CI], 88.9 to 100.0%) and a specificity of 100.0% (95% CI, 99.5 to 100.0%). The results of this study suggest that the ultrasensitive p24 antigen assay may be performed on capillary plasma stored on filter paper (sensitivity and specificity, 100.0%; n = 87) instead of venous plasma (sensitivity, 92.3%; specificity, 100.0%; n = 150). The use of glucolets (instruments used to perform capillary phlebotomies), instead of syringes and needles, may reduce procedural pain and the risk of needle stick injuries at a comparable cost. Compared to the reference, HIV could have been correctly excluded based on one rapid test for at least 90% of these children. The results of this study point towards underutilized opportunities to simplify phlebotomy and pediatric HIV screening.     

Diagnostic accuracy of rapid antigen tests in cerebrospinal fluid for pneumococcal meningitis: a systematic review and meta-analysis

BackgroundStreptococcus pneumoniae is a leading cause of bacterial meningitis worldwide. Conventional microbiological assays take several days and require the use of various drugs for empirical treatment. Rapid antigen tests in cerebrospinal fluid (CSF) may be useful to triage pneumococcal meningitis immediately.ObjectivesTo elucidate whether rapid antigen tests in CSF are useful in the triage of pneumococcal meningitis.MethodsData sourcesCochrane CENTRAL, MEDLINE, EMBASE, World Health Organization International Clinical Trials Registry Platform, and ClinicalTrials.gov databases were searched.Study eligibility criteriaAll types of cohort studies except multiple-group studies, where the sensitivity and specificity of rapid antigen tests in CSF compared with CSF culture can be extracted.ParticipantsPatients with suspected meningitis.TestsRapid antigen tests in CSF.Reference standardsOne or more of the following: blood culture, CSF culture, and polymerase chain reaction in CSF.Assessment of risk of biasThe methodological quality of the included studies was assessed using QUADAS-2.Methods of data synthesisWe used a random-effects bivariate model for the meta-analysis. We conducted a subgroup analysis by dividing studies into types of antigen tests, adults and children, low-income and high-income countries, and with or without exposure to antibiotics before lumbar puncture.ResultsForty-four studies involving 14 791 participants were included. Most studies had a moderate-to-low methodological quality. Summary sensitivity and specificity were 99.5% (95% confidence interval (CI), 92.4–100%) and 98.2% (95% CI, 96.9–98.9%), respectively. Positive predictive values and negative predictive values at the median prevalence (4.2%) in the included studies were 70.8% (95% CI, 56.6–79.9%) and 100% (95% CI, 99.7–100%), respectively. The diagnostic accuracy was consistent across the various subgroups, except for slightly reduced sensitivity in high-income countries.ConclusionsRapid antigen tests in CSF would be useful in triaging pneumococcal meningitis. Further studies are warranted to investigate the clinical benefit of ruling out pneumococcal meningitis based on the results of rapid antigen tests.     

Combining Parasite Lactate Dehydrogenase-Based and Histidine-Rich Protein 2-Based Rapid Tests To Improve Specificity for Diagnosis of Malaria Due to Plasmodium knowlesi and Other Plasmodium Species in Sabah,Malaysia

Plasmodium knowlesi causes severe and fatal malaria in Malaysia. Microscopic misdiagnosis is common and may delay appropriate treatment. P. knowlesi can cross-react with “species-specific” parasite lactate dehydrogenase (pLDH) monoclonal antibodies used in rapid diagnostic tests (RDTs) to detect P. falciparum and P. vivax. At one tertiary-care hospital and two district hospitals in Sabah, we prospectively evaluated two combination RDTs for malaria diagnosis by using both a pan-Plasmodium-pLDH (pan-pLDH)/P. falciparum-specific-pLDH (Pf-pLDH) RDT (OptiMAL-IT) and a non-P. falciparum VOM-pLDH/Pf-HRP2 RDT (CareStart). Differential cross-reactivity among these combinations was hypothesized to differentiate P. knowlesi from other Plasmodium monoinfections. Among 323 patients with PCR-confirmed P. knowlesi (n = 193), P. falciparum (n = 93), and P. vivax (n = 37) monoinfections, the VOM-pLDH individual component had the highest sensitivity for nonsevere (35%; 95% confidence interval [CI], 27 to 43%) and severe (92%; CI, 81 to 100%) P. knowlesi malaria. CareStart demonstrated a P. knowlesi sensitivity of 42% (CI, 34 to 49%) and specificity of 74% (CI, 65 to 82%), a P. vivax sensitivity of 83% (CI, 66 to 93%) and specificity of 71% (CI, 65 to 76%), and a P. falciparum sensitivity of 97% (CI, 90 to 99%) and specificity of 99% (CI, 97 to 100%). OptiMAL-IT demonstrated a P. knowlesi sensitivity of 32% (CI, 25 to 39%) and specificity of 21% (CI, 15 to 29%), a P. vivax sensitivity of 60% (CI, 42 to 75%) and specificity of 97% (CI, 94 to 99%), and a P. falciparum sensitivity of 82% (CI, 72 to 89%) and specificity of 39% (CI, 33 to 46%). The combination of CareStart plus OptiMAL-IT for P. knowlesi using predefined criteria gave a sensitivity of 25% (CI, 19 to 32%) and specificity of 97% (CI, 92 to 99%). Combining two RDT combinations was highly specific for P. knowlesi malaria diagnosis; however, sensitivity was poor. The specificity of pLDH RDTs was decreased for P. vivax and P. falciparum because of P. knowlesi cross-reactivity and cautions against their use alone in areas where P. knowlesi malaria is endemic. Sensitive P. knowlesi-specific RDTs and/or alternative molecular diagnostic tools are needed in areas where P. knowlesi malaria is endemic.     

Self-testing for the detection of SARS-CoV-2 infection with rapid antigen tests for people with suspected COVID-19 in the community

ObjectivesTo evaluate the performance of nasal mid-turbinate self-testing using rapid antigen detection tests (RDT) for persons with suspected coronavirus disease 2019 (COVID-19) in the community. Self-testing for COVID-19 infection with lateral flow assay severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RDT, provides rapid results and could enable frequent and extensive testing in the community, thereby improving the control of SARS-CoV-2.MethodsParticipants visiting a municipal SARS-CoV-2 testing centre, received self-testing kits containing either the BD Veritor System (BD-RDT) or Roche SARS-CoV-2 antigen detection test (Roche-RDT). Oro-nasopharyngeal swabs were collected from the participants for quantitative RT-PCR (qRT-PCR) testing. As a proxy for contagiousness, viral culture was performed on a selection of qRT-PCR positive samples to determine the Ct-value at which the chance of a positive culture dropped below 0.5 (Ct-value cut-off). Sensitivity and specificity of self-testing were compared to qRT-PCR with a Ct-value below the Ct value cut-off. Determinants independently associated with a false-negative self-test result were determined.ResultsA total of 3201 participants were included (BD-RDT n = 1595; Roche-RDT n = 1606). Sensitivity and specificity of self-testing compared with the qRT-PCR results with a Ct-value below the Ct-value cut-off were 78.4% (95% CI 73.2%–83.5%) and 99.4% (95% CI 99.1%–99.7%), respectively. A higher age was independently associated with a false-negative self-testing result with an odds ratio of 1.024 (95% CI 1.003–1.044).ConclusionsSelf-testing using currently available RDT has a high specificity and relatively high sensitivity to identify individuals with a high probability of contagiousness.     

Clinical and Analytical Performance of the Onclarity HPV Assay Using the VALGENT Framework

As the demand for human papillomavirus (HPV)-related cervical screening increases, emerging HPV tests must be evaluated robustly using well-annotated samples, such as those generated in the Validation of HPV Genotyping Tests (VALGENT) framework. Through VALGENT, we assessed the performance of the BD Onclarity HPV assay, which detects 14 high-risk (HR) types and resolves six individual types and three groups of types. Consecutive samples from a screening population (n = 1,000), enriched with cytologically abnormal samples (n = 300), that had been tested previously with the GP5+/6+ PCR enzyme immunoassay (EIA) and the GP5+/6+ PCR LMNX assay (Diassay) were tested with the Onclarity assay. Type-specific HPV prevalences were analyzed according to age and cytological result. The accuracy of the Onclarity assay for the detection of cervical intraepithelial neoplasia grade 2+ (CIN2+) and CIN3+ was assessed relative to the GP5+/6+ EIA results by using noninferiority criteria. Overall agreement and type-specific agreement between the Onclarity assay and the GP5+/6+ LMNX assay were assessed. The prevalence of HPV types 16, 18, 31, and 45 increased with the severity of cytological results (P for trend, <0.05). For the detection of CIN2+, the Onclarity assay had a relative sensitivity of 1.02 (95% confidence interval [CI], 0.99 to 1.05; P < 0.001 for noninferiority) and a relative specificity of 0.99 (95% CI, 0.97 to 1.00; P = 0.186 for noninferiority). The kappa for agreement between the Onclarity assay and the GP5+/6+ LMNX assay for HR-HPV was 0.92 (95% CI, 0.89 to 0.94), and values for the six individual types ranged from 0.78 (95% CI, 0.68 to 0.87) for HPV-52 to 0.96 (95% CI, 0.93 to 0.99) for HPV-16. These data suggest that the Onclarity assay offers applications for clinical workstreams while providing genotyping information that may be useful for risk stratification beyond types 16 and 18.     

Performance of SaSelect,a Chromogenic Medium for Detection of Staphylococci in Clinical Specimens

In a preliminary study, known staphylococcus (n = 86) and other microbial (n = 12) isolates were plated on three chromogenic media, SaSelect (Bio-Rad, Hercules, CA, USA), CHROMagar Staph. aureus (CHROMagar Microbiology, Paris, France), and S. aureus ID (bioMérieux, Marcy l''Etoile, France). The sensitivities of all the media to detect Staphylococcus aureus after 24 h of incubation were high (100.0%). However, their specificities varied at 93.3% (95% confidence interval [CI], 86.0% to 100.0%) (CHROMagar Staph. aureus), 97.8% (95% CI, 93.5% to 100.0%) (S. aureus ID), and 100.0% (SaSelect). SaSelect also showed the highest sensitivity for recovery and differentiation of other staphylococci. As the best performing chromogenic medium, SaSelect was then prospectively compared to conventional culture and identification tests for the detection of staphylococci from 2,780 clinical specimens. A total of 1,589 staphylococcal isolates were recovered. Of these, 912 were S. aureus and 677 were other staphylococci. The sensitivity and specificity of SaSelect to detect S. aureus in clinical specimens after 24 h of incubation were 99.6% and 99.9% (95% CI, 99.2% to 100.0% and 99.8% to 100.0%), respectively, whereas the sensitivity and specificity using conventional plates combined with laboratory identification methods were 96.8% and 99.5% (95% CI, 95.7 to 97.9% and 99.2% to 99.8%). For the recovery and preliminary identification of other staphylococci, the sensitivity and specificity of SaSelect were 94.4% and 99.9%. SaSelect is a well-performing chromogenic medium that significantly improved the detection of staphylococci, especially S. aureus, compared to conventional culture (P < 0.0001).     

Validation of a Fluorescence In Situ Hybridization Method Using Peptide Nucleic Acid Probes for Detection of Helicobacter pylori Clarithromycin Resistance in Gastric Biopsy Specimens

Here, we evaluated a previously established peptide nucleic acid-fluorescence in situ hybridization (PNA-FISH) method as a new diagnostic test for Helicobacter pylori clarithromycin resistance detection in paraffin-embedded gastric biopsy specimens. Both a retrospective study and a prospective cohort study were conducted to evaluate the specificity and sensitivity of a PNA-FISH method to determine H. pylori clarithromycin resistance. In the retrospective study (n = 30 patients), full agreement between PNA-FISH and PCR-sequencing was observed. Compared to the reference method (culture followed by Etest), the specificity and sensitivity of PNA-FISH were 90.9% (95% confidence interval [CI], 57.1% to 99.5%) and 84.2% (95% CI, 59.5% to 95.8%), respectively. In the prospective cohort (n = 93 patients), 21 cases were positive by culture. For the patients harboring clarithromycin-resistant H. pylori, the method showed sensitivity of 80.0% (95% CI, 29.9% to 98.9%) and specificity of 93.8% (95% CI, 67.7% to 99.7%). These values likely represent underestimations, as some of the discrepant results corresponded to patients infected by more than one strain. PNA-FISH appears to be a simple, quick, and accurate method for detecting H. pylori clarithromycin resistance in paraffin-embedded biopsy specimens. It is also the only one of the methods assessed here that allows direct and specific visualization of this microorganism within the biopsy specimens, a characteristic that allowed the observation that cells of different H. pylori strains can subsist in very close proximity in the stomach.     

Accuracy of cholera rapid diagnostic tests: a systematic review and meta-analysis

BackgroundCholera is an acute diarrheal disease caused by Vibrio cholerae O1 or O139. Cholera rapid diagnostic tests (RDTs) are widely used to screen for cholera cases. However, their accuracy has not been systematically reviewed.ObjectivesTo evaluate the diagnostic accuracy of cholera RDTs.MethodsSystematic review and meta-analysis.Data sourcesMedline, EMBASE and Web of science through to November 2020; references of included studies and a technical guidance on cholera RDTs. This review is registered with PROSPERO (CRD42021233124).Study eligibility criteriaCross-sectional studies comparing the performance of cholera RDTs either to stool culture or PCR.ParticipantsIndividuals with clinically suspected cholera.Data extractionTwo authors independently extracted data and assessed the quality using Quality Assessment of Diagnostic Accuracy Studies 2 (QUADAS-2) criteria.ResultsEighteen studies were included in the systematic review of which 17 were used for meta-analysis. Crystal VC was the most frequently used RDT (13 studies), followed by Cholkit and Institut Pasteur cholera dipstick (three studies each), SD Bioline (two studies), Artron (one study) and Smart (one study). Using direct testing (n = 12 627 specimens), the bivariate random-effects model yielded a pooled sensitivity and specificity of 91% (95% CI 87%–94%) and 80% (95% CI 74%–84%), respectively. However, through alkaline peptone water (APW) enrichment (n = 3403 specimens), the pooled sensitivity and specificity were 89% (95% CI 79%–95%) and 98% (95% CI 95%–99%), respectively.ConclusionCholera RDTs, especially when enriched with APW, have moderate sensitivity and specificity. Although less useful for clinical management, the current generation of RDTs have clear utility for surveillance efforts if used in a principled manner. Enrichment of stool specimens in APW before using cholera RDTs reduces the possibility of obtaining false-positive results, despite the few cholera cases that go undetected. It is noteworthy that APW-enriched cholera RDTs are not necessarily rapid tests, and are not listed in the Global Task Force on Cholera Control/WHO target product profile.