Extraction-Free Colorimetric RT-LAMP Detection of SARS-CoV-2 in Saliva.

The pandemic situation caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has highlighted the need of fast, simple, and cost-effective tests for the diagnosis of emerging pathogens. RT-qPCR has been established as the reference technique for the diagnosis of SARS-CoV-2 infections. This method requires a time-consuming protocol for the extraction of the nucleic acids present in the sample. A colorimetric reverse transcription loop-mediated isothermal amplification using the calcein molecule combined with a simple extraction-free method for saliva samples (calcein RT-LAMP) has been developed. Samples are heated 95 °C for 10 min before amplification at 63 °C for 40 min. The results can be observed by fluorescence or by the naked eye with a color change from orange to green. The method was compared with commercialized available colorimetric and fluorescent RT-LAMP kits. The developed method shows better sensitivity and specificity than the colorimetric commercial RT-LAMP and the same as the fluorescent RT-LAMP, without the need of a fluorescent reader. Moreover, the calcein RT-LAMP has, compared to RT-qPCR, a sensitivity of 90% and a specificity of 100% for saliva samples with a Ct ≤ 34, without the need for expensive RT-qPCR instruments, demonstrating the potential of this method for population screening.

Enhancing SARS-CoV-2 Surveillance through Regular Genomic Sequencing in Spain: The RELECOV Network.

Millions of SARS-CoV-2 whole genome sequences have been generated to date. However, good quality data and adequate surveillance systems are required to contribute to meaningful surveillance in public health. In this context, the network of Spanish laboratories for coronavirus (RELECOV) was created with the main goal of promoting actions to speed up the detection, analyses, and evaluation of SARS-CoV-2 at a national level, partially structured and financed by an ECDC-HERA-Incubator action (ECDC/GRANT/2021/024). A SARS-CoV-2 sequencing quality control assessment (QCA) was developed to evaluate the network's technical capacity. QCA full panel results showed a lower hit rate for lineage assignment compared to that obtained for variants. Genomic data comprising 48,578 viral genomes were studied and evaluated to monitor SARS-CoV-2. The developed network actions showed a 36% increase in sharing viral sequences. In addition, analysis of lineage/sublineage-defining mutations to track the virus showed characteristic mutation profiles for the Delta and Omicron variants. Further, phylogenetic analyses strongly correlated with different variant clusters, obtaining a robust reference tree. The RELECOV network has made it possible to improve and enhance the genomic surveillance of SARS-CoV-2 in Spain. It has provided and evaluated genomic tools for viral genome monitoring and characterization that make it possible to increase knowledge efficiently and quickly, promoting the genomic surveillance of SARS-CoV-2 in Spain.

Validation of Rapid and Economic Colorimetric Nanoparticle Assay for SARS-CoV-2 RNA Detection in Saliva and Nasopharyngeal Swabs.

Even with the widespread uptake of vaccines, the SARS-CoV-2-induced COVID-19 pandemic continues to overwhelm many healthcare systems worldwide. Consequently, massive scale molecular diagnostic testing remains a key strategy to control the ongoing pandemic, and the need for instrument-free, economic and easy-to-use molecular diagnostic alternatives to PCR remains a goal of many healthcare providers, including WHO. We developed a test (Repvit) based on gold nanoparticles that can detect SARS-CoV-2 RNA directly from nasopharyngeal swab or saliva samples with a limit of detection (LOD) of 2.1 × 105 copies mL-1 by the naked eye (or 8 × 104 copies mL-1 by spectrophotometer) in less than 20 min, without the need for any instrumentation, and with a manufacturing price of <$1. We tested this technology on 1143 clinical samples from RNA extracted from nasopharyngeal swabs (n = 188), directly from saliva samples (n = 635; assayed by spectrophotometer) and nasopharyngeal swabs (n = 320) from multiple centers and obtained sensitivity values of 92.86%, 93.75% and 94.57% and specificities of 93.22%, 97.96% and 94.76%, respectively. To our knowledge, this is the first description of a colloidal nanoparticle assay that allows for rapid nucleic acid detection at clinically relevant sensitivity without the need for external instrumentation that could be used in resource-limited settings or for self-testing.

Validation of a rapid, saliva-based, and ultra-sensitive SARS-CoV-2 screening system for pandemic-scale infection surveillance.

Without any realistic prospect of comprehensive global vaccine coverage and lasting immunity, control of pandemics such as COVID-19 will require implementation of large-scale, rapid identification and isolation of infectious individuals to limit further transmission. Here, we describe an automated, high-throughput integrated screening platform, incorporating saliva-based loop-mediated isothermal amplification (LAMP) technology, that is designed for population-scale sensitive detection of infectious carriers of SARS-CoV-2 RNA. Central to this surveillance system is the "Sentinel" testing instrument, which is capable of reporting results within 25 min of saliva sample collection with a throughput of up to 3840 results per hour. It incorporates continuous flow loading of samples at random intervals to cost-effectively adjust for fluctuations in testing demand. Independent validation of our saliva-based RT-LAMP technology on an automated LAMP instrument coined the "Sentinel", found 98.7% sensitivity, 97.6% specificity, and 98% accuracy against a RT-PCR comparator assay, confirming its suitability for surveillance screening. This Sentinel surveillance system offers a feasible and scalable approach to complement vaccination, to curb the spread of COVID-19 variants, and control future pandemics to save lives.

Impact of surface topography on the bacterial attachment to micro- and nano-patterned polymer films.

The development of antimicrobial surfaces has become a high priority in recent times. There are two ongoing worldwide health crises: the COVID-19 pandemic provoked by the SARS-CoV-2 virus and the antibiotic-resistant diseases provoked by bacteria resistant to antibiotic-based treatments. The need for antimicrobial surfaces against bacteria and virus is a common factor to both crises. Most extended strategies to prevent bacterial associated infections rely on chemical based-approaches based on surface coatings or biocide encapsulated agents that release chemical agents. A critical limitation of these chemistry-based strategies is their limited effectiveness in time while grows the concerns about the long-term toxicity on human beings and environment pollution. An alternative strategy to prevent bacterial attachment consists in the introduction of physical modification to the surface. Pursuing this chemistry-independent strategy, we present a fabrication process of surface topographies [one-level (micro, nano) and hierarchical (micro+nano) structures] in polypropylene (PP) substrates and discuss how wettability, topography and patterns size influence on its antibacterial properties. Using nanoimprint lithography as patterning technique, we report as best results 82 and 86% reduction in the bacterial attachment of E. coli and S. aureus for hierarchically patterned samples compared to unpatterned reference surfaces. Furthermore, we benchmark the mechanical properties of the patterned PP surfaces against commercially available antimicrobial films and provide evidence for the patterned PP films to be suitable candidates for use as antibacterial functional surfaces in a hospital environment.

Comparison of Four Methods for Streptococcus pneumoniae Capsular Typing.

BACKGROUND: Pneumococcal capsular-type identification is essential for monitoring the epidemiology of pneumococcal infections and for establishing the effectiveness of pneumococcal vaccines. The objective of this work was to compare the accuracy of four methods of Streptococcus pneumoniae capsular typing. METHODS: A prospective blind study was carried out at Donostia University Hospital (northern Spain) to determine the capsular types of 50 pneumococcal clinical isolates using four techniques: a) S. PneumoStripTM: a reverse-hybridization strip-based commercial assay that detects 76 pneumococcal serotypes: 42 individually and 34 in pairs. b) FAF-mPCR: a single-step multiplex-PCR (polymerase chain reaction) assay combined with fragment analysis using automated fluorescent capillary electrophoresis, which can differentiate 92 serotypes in a single tube: 31 individually, 28 in pairs, and 33 in groups of 3 to 5 serotypes. c) PCRSeqTyping: which enables the detection of 91 serotypes after sequencing the regions of the cpsB gene in two steps: 59 directly and the remaining 32 serotypes in a second step. d) The Quellung reaction. RESULTS: The S. PneumoStripTM, FAF-mPCR and PCRSeqTyping identified the serotypes of all the 50 clinical isolates. With the Quellung reaction 46/50 (92%) isolates were correctly serotyped. The quickest technique was the S. PneumoStripTM, followed by the single-step multiplex PCR assay and PCRSeqTyping. The Quellung reaction was the slowest technique. CONCLUSIONS: The S. PneumoStripTM, PCRSeqTyping, and FAF-mPCR were very accurate techniques for pneumococcal serotyping, with S. PneumoStripTM obtaining results more rapidly. The combination of any of these S. pneumoniae molecular typing techniques and the Quellung reaction as confirmation reference method is a highly precise and fast strategy for the serotyping of high number of pneumococcal clinical isolates.

Fatal Case of Nosocomial Legionella pneumophila Pneumonia, Spain, 2018.

A nosocomial case of Legionella pneumophila pneumonia likely caused by a serogroup 3 strain was detected by a urinary antigen test in Spain in 2018. Although Legionella bacteria could not be isolated from respiratory samples, molecular methods implicated the sink faucet of the patient's room as the probable infection source.

Variants of the bile: solubility test to differentiate Streptococcus pneumoniae from other viridans group streptococci.

Aim: Bile salts promote the specific autolysis of pneumococcal cells, allowing the differentiation between Streptococcus pneumoniae and other viridans group streptococci (VGS). Material & methods: One hundred clinical VGS isolates identified by amplification and sequencing of 16S rRNA, groEL and sodA genes were analyzed with different variants of bile-solubility tests: tube testing read by naked eye; tube testing where the lysis was measured as the decrease of turbidity with a densitometer; and direct testing on blood agar plate. Results: As expected, all S. pneumoniae isolates were fully lysed in the presence of bile salts except for one isolate that partially lysate in tube testing as well as on the blood agar plate. None of the VGS were lysed by bile salts. Conclusion: Bile-solubility testing is an accurate and technically nondemanding method to discriminate between S. pneumoniae and other VGS species.

Primary Cutaneous Nocardia brasiliensis in a Spanish Child.

BACKGROUND: We report a case of a primary cutaneous nocardiosis by autochthonous Nocardia brasiliensis in a Spanish immunocompetent 9-year-old boy. METHODS: N. brasiliensis caused cellulitis showing the patient recovery after drainage and treatment with trimethoprim-sulfamethoxazole. Nocardia grew in pure culture and its identification was confirmed by sequencing (16S rRNA) and by MALDI-TOF MS (Bruker, Daltonics, Germany). CONCLUSIONS: In Spain although N. brasiliensis cutaneous infections in children are very infrequent should not be ruled out when an insect bite, stuck with a pine needle or an animal scratch has existed and the wound evolution is torpid.

A reverse-hybridization test for the identification of 76 pneumococcal serotypes, 42 individually and 34 in pairs.

The S. PneumoStrip test is a recently developed reverse hybridization strip-based commercial assay that allows for the identification of 76 pneumococcal serotypes, 42 individually and 34 in pairs, according to their specific gene sequences. The test was validated with reference strains of 92 different pneumococcal serotypes and with a selection of 75 clinical isolates representing 55 serotypes, showing 100% sensitivity and specificity. The test was also applied to 64 pneumococcal invasive isolates (23 different serotypes) consecutively collected between June 2016 and March 2017, with 60 (93.8%) being serotyped. Four isolates belonging to serotypes 13, 29, and 35B (2 isolates), which are not included in the test, did not produce a hybridization signal with serotype specific probes. The identification of most serotypes causing invasive pneumococcal disease together with the simplicity of performance and results interpretation, and the use of routine laboratory equipment make this test very suitable for most clinical and research laboratories.

Single-Step Multiplex PCR Assay for Determining 92 Pneumococcal Serotypes.

For pneumococcal disease surveillance, simple and cost-effective methods capable of determining all serotypes are needed. Combining a single-tube multiplex PCR with fluorescently labeled primers followed by amplicon analysis using automated fluorescent capillary electrophoresis, each serotype of 92 reference isolates and 297 recently collected clinical isolates was successfully determined.

Long-Term Epidemiology of Streptococcus pneumoniae Serogroup 6 in a Region of Southern Europe with Special Reference to Serotype 6E.

Streptococcus pneumoniae serotype 6E has recently been described, but its long-term epidemiology is not well known. From 1981-2013, 704 serogroup 6 clinical isolates were obtained in Gipuzkoa, Basque Country, Spain. All invasive and one in four non-invasive isolates were included. Overall, 75, 97, 51 and 45 serotypes 6A, 6B, 6C and 6E isolates, respectively, were detected. No serotype 6D isolates were identified. The prevalence of serotypes 6E and 6B, but not that of serotypes 6A and 6C, declined after the introduction of pneumococcal conjugate vaccines. Serotype 6E isolates showed the highest resistance rate. Most serotype 6E isolates were ST90.

Progressive decrease in the potential usefulness of meningococcal serogroup B vaccine (4CMenB, Bexsero®) in Gipuzkoa, Northern Spain.

The effectiveness of a vaccine is determined not only by the immunogenicity of its components, but especially by how widely it covers the disease-causing strains circulating in a given region. Because vaccine coverage varies over time, this study aimed to detect possible changes that could affect vaccine protection during a specific period in a southern European region. The 4CMenB vaccine is licensed for use in Europe, Canada, and Australia and is mainly directed against Neisseria meningitidis serogroup B. This vaccine contains four main immunogenic components: three recombinant proteins, FHbp, Nhba and NadA, and an outer membrane vesicle [PorA P1.4]. The allelic distribution of FHbp, Nhba, NadA, and PorA antigens in 82 invasive isolates (B and non-B serogroups) isolated from January 2008 to December 2013 were analyzed. 4CMenB was likely protective against 61.8% and 50% of serogroup B and non-B meningococci, respectively, in the entire period, but between 2012 and 2013, the predicted protection fell below 45% (42.1% for serogroup B isolates).The observed decreasing trend in the predicted protection during the 6 years of the study (Χ2 for trend  = 4.68, p = 0.03) coincided with a progressive decrease of several clonal complexes (e.g., cc11, cc32 and cc41/44), which had one or more antigens against which the vaccine would offer protection.

Two outbreaks of Listeria monocytogenes infection, Northern Spain.

In the province of Gipuzkoa, Spain (≈700,000 inhabitants), 7-12 episodes of human listeriosis were recorded annually during 2009-2012. However, during January 2013-February 2014, 27 episodes were detected, including 11 pregnancy-associated cases. Fifteen cases in 2 epidemiologically unrelated outbreaks were caused by a rare type of Listeria monocytogenes, sequence type 87 serotype 1/2b.

Dynamics of Streptococcus pneumoniae serotypes causing acute otitis media isolated from children with spontaneous middle-ear drainage over a 12-year period (1999-2010) in a region of northern Spain.

The aim of this study was to determine the serotype and clonal distribution of pneumococci causing acute otitis media (AOM) and their relationship with recurrences and mixed infections with other microorganisms under the influence of the 7-valent pneumococcal conjugate vaccine (PCV7). To do this, all pneumococcal isolates collected from the spontaneous middle-ear drainage of children <5 years old diagnosed of AOM by their pediatrician or their general practitioner from 1999 to 2010 were phenotypically characterized and the most frequent serotypes were genotyped. In the 12-year study, 818 episodes of pneumococcal AOM were detected, mostly (70.5%) in children younger than 2 years old. In 262 episodes (32%), the pneumococci were isolated with another bacterium, mainly (n=214) Haemophilus influenzae. Mixed infections were similar in children under or over 2 years old. The most frequent serotypes were 19A (n=227, 27.8%), 3 (n=92, 11.2%) and 19F (n=74, 9%). Serotypes included in the PCV7 sharply decreased from 62.4% in the pre-vaccination (1999-2001) to 2.2% in the late post-vaccination period (2008-2010). Serotype diversity steadily increased after the introduction of the PCV7 but decreased from 2008-2010 due to the predominant role of serotype 19A isolates, mostly ST276 and ST320. The prevalence of serotype 3 doubled from 6.1% (20/326) in 1999-2004 to 14.6% (72/492) in 2005-2010. Relapses mainly occurred in male infants infected with isolates with diminished antimicrobial susceptibility. Reinfections caused by isolates with the same serotype but different genotype were frequent, highlighting the need for genetic studies to differentiate among similar strains. In conclusion, the main change in pneumococcal AOM observed after the introduction of the PCV7 was the sharp decrease in vaccine serotypes. Also notable was the high burden of serotype 19A in total pneumococcal AOM before and especially after the introduction of the PCV7, as well as in relapses and reinfections.

Decline and rise of the antimicrobial susceptibility of Streptococcus pneumoniae isolated from middle ear fluid in children: influence of changes in circulating serotypes.

Changes in the antimicrobial susceptibility of Streptococcus pneumoniae causing otitis media were studied in 916 isolates from children <5 years old between 1999 and 2010 in a region of northern Spain. The rate of antimicrobial resistance decreased between the period before the introduction of the heptavalent pneumococcal conjugate vaccine (from 1999 to 2001) and the period from 2005 to 2007. However, in 2008 to 2010, resistance rates increased again due to the spread of serotype 19A, especially the multidrug-resistant ST320 and ST276 clones.

Dynamics of pneumococcal nasopharyngeal carriage in healthy children attending a day care center in northern Spain. Influence of detection techniques on the results.

BACKGROUND: Pneumococcal nasopharyngeal carriage precedes invasive infection and is the source for dissemination of the disease. Differences in sampling methodology, isolation or identification techniques, as well as the period (pre -or post-vaccination) when the study was performed, can influence the reported rates of colonization and the distribution of serotypes carried. OBJECTIVES: To evaluate the prevalence and dynamics of pneumococcal nasopharyngeal colonization in healthy children aged 6-34 months attending a day care center with a high level of hygiene and no overcrowding. The study was performed 3-4 years after the 7-valent pneumococcal vaccine was introduced, using multiple methodologies to detect and characterize the isolates. METHODS: Over 12 months, 25 children were sampled three times, 53 children twice and 27 children once. Three Streptococcus pneumoniae typing techniques were used: Quellung, Pneumotest-Latex-kit and multiplex-polymerase chain reaction (PCR). The similarity of isolates of the same serotype was established by pulsed field gel electrophoresis (PFGE) and occasionally the multilocus sequence type (ST) was also determined. RESULTS: Overall pneumococcal carriage and multiple colonization rates were 89.5% (94/105) and 39%, respectively. Among 218 pneumococci detected, 21 different serotypes and 13 non-typeable isolates were found. The most prevalent serotypes were 19A, 16F and 15B. Serotypes 15B, 19A and 21 were mainly found as single carriage; in contrast serotypes 6B, 11A and 20, as well as infrequent serotypes, were isolated mainly as part of multiple carriage. Most 19A isolates were ST193 but most serotypes showed high genetic heterogeneity. Changes in the pneumococci colonizing each child were frequent and the same serotype detected on two occasions frequently showed a different genotype. By multiplex-PCR, 100% of pneumococci could be detected and 94% could be serotyped versus 80.3% by the Quellung reaction and Pneumotest-Latex in combination (p < 0.001). CONCLUSIONS: Rates of S. pneumoniae carriage and multiple colonization were very high. Prevalent serotypes differed from those found in similar studies in the pre-vaccination period. In the same child, clearance of a pneumococcal strain and acquisition of a new one was frequent in a short period of time. The most effective technique for detecting pneumococcal nasopharyngeal carriers was multiplex-PCR.

A novel Real Time micro PCR based Point-of-Care device for Salmonella detection in human clinical samples.

Our POC (Point of Care) device is intended to be a diagnostic tool for routine use in the clinical sector. The validation of the whole procedure, including bacterial genomic DNA isolation and the Real Time detection of Salmonella spp., was conducted on 29 clinical stool samples that had been diagnosed with Salmonella spp. by a routine culture technique. The entire process was achieved in a single microfluidic chip within 35 min. In comparison to the culture reference method that is used in the clinical laboratories, this new device performed well in regards to the analytical parameters of sensitivity, specificity and accuracy. Therefore, the POC device reported in this study proved to be very appropriate for the fully integrated analysis system. To the best of our knowledge, this is the first work to report the sample preparation and followed by Real Time PCR (Polymerase Chain Reaction) on a single 2.5 µl chamber chip for the detection of Salmonella spp. bacteria in stool samples.

Molecular characterization of Staphylococcus aureus carrying the panton-valentine leucocidin genes in northern Spain.

OBJECTIVES: To study the prevalence of the Panton-Valentine leucocidin (PVL) gene in methicillin-susceptible (MSSA) and methicillin-resistant (MRSA) Staphylococcus aureus obtained in Gipuzkoa, northeastern area of the Basque Country, north-central Spain, and perform the molecular characterization of PVL-positive isolates. METHODS: Molecular studies comprised: PVL gene detection by PCR, staphylococcal chromosome cassette mec (SCCmec) typing, spa sequencing, multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE), and detection of the arginine catabolic mobile element (ACME). RESULTS: Between 1978 and 2006, only two (0.3%) of the 686 MRSA isolates studied were positive for the PVL gene. This percentage increased between 2007 and 2009, when the PVL gene was detected in 30 of the 679 MRSA (4.4%) and in nine of the 1227 MSSA (0.7%) isolates. The 41 PVL-positive isolates characterized had eight different sequence types (STs). Twenty-three MRSA PVL-positive isolates were ST8, spa type t008, seven of which were ACME positive, erythromycin-resistant and showed the PFGE pattern (90-100% similarity) of the USA300 clone. ST8 was also the most prevalent ST among the nine MSSA PVL-positive isolates. CONCLUSION: The current epidemiology of PVL-positive MRSA in our region more closely resembles that of the USA rather than that of other European countries, being USA300 or USA300-like isolates the most prevalent ones.

Antimicrobial susceptibilities and serotypes of Streptococcus pneumoniae isolates from elderly patients with pneumonia and acute exacerbation of chronic obstructive pulmonary disease.

In the elderly, Streptococcus pneumoniae is the most common cause of pneumonia and one of the most frequently isolated pathogens in cases of acute exacerbation of chronic obstructive pulmonary disease (AECOPD). This study was conducted to compare the pneumococcal isolates obtained during episodes of AECOPD and pneumonia in patients of ≥65 years old and to analyze whether in patients with AECOPD and pneumonia within a short interval, the same isolate caused both episodes. This laboratory-based study was performed between 2005 and 2008. Pneumococcal isolates from episodes of pneumonia (n = 401) and AECOPD (n = 398), matched one-to-one by date of isolation, were characterized. The serotypes and genotypes of other pneumococcal isolates causing pneumonia and AECOPD in the same patient were compared. In patients with pneumonia, COPD as an underlying disease was not associated with more-drug-resistant pneumococci. In contrast, isolates causing AECOPD showed higher rates of resistance than those causing pneumonia. Serotypes 1, 3, and 7F were more frequent in pneumonia. The same pneumococcus was involved in 25.7% (9/35 patients) of patients with two consecutive AECOPD episodes but in only 6.3% (2/32 patients) of COPD patients with pneumonia and exacerbation (Fisher's exact test; P = 0.047). Less invasive serotypes were isolated more often in AECOPD and were more resistant to antimicrobials. The presence of a specific pneumococcal serotype in AECOPD does not predict the etiology of subsequent pneumonia.