ABSTRACT
[This corrects the article DOI: 10.1016/j.heliyon.2021.e06328.].
ABSTRACT
Possible pre- or asymptomatic transmission has been reported, both from SARS-CoV and from MERS-CoV outbreaks, although this appears to be uncommon. In contrast, during the COVID-19 pandemic, an increasing number of studies and case reports indicate that pre- or asymptomatic transmission of SARS-CoV-2 is not only possible but also occurs frequently. We report repeated rRT-PCR detection of SARS-CoV-2 in a health care worker and demonstrate infective ability up to three days prior to mild COVID-19 symptoms. rRT-PCR indicated high viral levels approximately three days after exposure. Viral samples collected one and three days prior to symptoms exhibited infectivity on Vero E6 cells, confirmed by detection of double-stranded RNA by immunofluorescence, assessment of cytopathic effect (CPE) and rRT-PCR. SARS-CoV-2 specific IgM and IgG antibodies were detected by day 9 and 15, respectively, after symptom onset. We propose that this provides evidence for potential early presymptomatic transmission of SARS-CoV-2 and that infectivity may be manifest shortly after exposure.
ABSTRACT
Here, we report a case of COVID-19-related acute necrotizing encephalopathy where SARS-CoV-2 RNA was found in CSF 19 days after symptom onset after testing negative twice. Although monocytes and protein levels in CSF were only marginally increased, and our patient never experienced a hyperinflammatory state, her neurologic function deteriorated into coma. MRI of the brain showed pathologic signal symmetrically in central thalami, subinsular regions, medial temporal lobes, and brain stem. Extremely high concentrations of the neuronal injury markers neurofilament light and tau, as well as an astrocytic activation marker, glial fibrillary acidic protein, were measured in CSF. Neuronal rescue proteins and other pathways were elevated in the in-depth proteomics analysis. The patient received IV immunoglobulins and plasma exchange. Her neurologic status improved, and she was extubated 4 weeks after symptom onset. This case report highlights the neurotropism of SARS-CoV-2 in selected patients and emphasizes the importance of repeated lumbar punctures and CSF analyses in patients with suspected COVID-19 and neurologic symptoms.
Subject(s)
Brain/diagnostic imaging , Coronavirus Infections/cerebrospinal fluid , Leukoencephalitis, Acute Hemorrhagic/cerebrospinal fluid , Pneumonia, Viral/cerebrospinal fluid , RNA, Viral/cerebrospinal fluid , Acyclovir/therapeutic use , Antiviral Agents/therapeutic use , Betacoronavirus/genetics , COVID-19 , Coronavirus Infections/diagnostic imaging , Coronavirus Infections/physiopathology , Coronavirus Infections/therapy , Female , Glial Fibrillary Acidic Protein/cerebrospinal fluid , Humans , Immunoglobulins, Intravenous/therapeutic use , Immunologic Factors/therapeutic use , Interleukin-6/cerebrospinal fluid , Leukoencephalitis, Acute Hemorrhagic/diagnostic imaging , Leukoencephalitis, Acute Hemorrhagic/physiopathology , Leukoencephalitis, Acute Hemorrhagic/therapy , Magnetic Resonance Imaging , Middle Aged , Neurofilament Proteins/cerebrospinal fluid , Pandemics , Plasma Exchange , Pneumonia, Viral/diagnostic imaging , Pneumonia, Viral/physiopathology , Pneumonia, Viral/therapy , Real-Time Polymerase Chain Reaction , SARS-CoV-2 , Tomography, X-Ray Computed , Viral Tropism , tau Proteins/cerebrospinal fluidABSTRACT
With the increasing pace of global warming, it is important to understand the role of meteorological factors in influenza virus (IV) epidemics. In this study, we investigated the impact of temperature, UV index, humidity, wind speed, atmospheric pressure, and precipitation on IV activity in Norway, Sweden, Finland, Estonia, Latvia and Lithuania during 2010â»2018. Both correlation and machine learning analyses revealed that low temperature and UV indexes were the most predictive meteorological factors for IV epidemics in Northern Europe. Our in vitro experiments confirmed that low temperature and UV radiation preserved IV infectivity. Associations between these meteorological factors and IV activity could improve surveillance and promote development of accurate predictive models for future influenza outbreaks in the region.
Subject(s)
Cold Temperature , Global Warming , Influenza, Human/epidemiology , Orthomyxoviridae/radiation effects , Ultraviolet Rays , Cell Line , Cell Survival , Cells, Cultured , Europe/epidemiology , Humans , Humidity , Macrophages/virology , Norway/epidemiology , Sweden/epidemiology , WindABSTRACT
Mycoplasma pneumoniae causes respiratory infections, such as community-acquired pneumonia (CAP), with epidemics recurring every 3 to 7 years. In 2010 and 2011, many countries experienced an extraordinary epidemic peak. The cause of these recurring epidemics is not understood, but decreasing herd immunity and shifts in the strains' antigenic properties have been suggested as contributing factors. M. pneumoniae PCR-positive samples were collected between 1996 and 2017 from four neighboring counties inhabited by 12% of Sweden's population. A total of 578 isolates were characterized directly from 624 clinical samples using P1 typing by sequencing and multilocus variable number tandem repeat analysis (MLVA). A fluorescence resonance energy transfer (FRET)-PCR approach was also used to detect mutations associated with macrolide resistance in the 23S rRNA gene. Through P1 typing, the strains were classified into type 1 and type 2, as well as variants 2a, 2b, 2c, and a new variant found in nine of the strains, denoted variant 2e. Twelve MLVA types were distinguished, and 3-5-6-2 (42.4%), 4-5-7-2 (37.4%), and 3-6-6-2 (14.9%) predominated. Several P1 and MLVA types cocirculated each year, but type 2/variant 2 strains and MLVA types 3-5-6-2 and 4-5-7-2 predominated during the epidemic period comprising the peak of 2010 and 2011. In 2016 and 2017, type 1 became more common, and MLVA type 4-5-7-2 predominated. We also found that 0.2% (1/578) of the strains carried a macrolide resistance-associated mutation, indicating a very low prevalence of macrolide resistance in this region of Sweden.
Subject(s)
Adhesins, Bacterial/genetics , Genetic Variation , Molecular Typing , Mycoplasma pneumoniae/classification , Mycoplasma pneumoniae/genetics , Pneumonia, Mycoplasma/epidemiology , Pneumonia, Mycoplasma/microbiology , Adhesins, Bacterial/chemistry , Adolescent , Adult , Aged , Aged, 80 and over , Amino Acid Sequence , Child , Child, Preschool , Female , History, 20th Century , History, 21st Century , Humans , Infant , Male , Middle Aged , Minisatellite Repeats , Multilocus Sequence Typing , Pneumonia, Mycoplasma/history , Sweden/epidemiology , Young AdultABSTRACT
According to the WHO, there is an urgent need for better control of viral diseases. Re-positioning existing safe-in-human antiviral agents from one viral disease to another could play a pivotal role in this process. Here, we reviewed all approved, investigational and experimental antiviral agents, which are safe in man, and identified 59 compounds that target at least three viral diseases. We tested 55 of these compounds against eight different RNA and DNA viruses. We found novel activities for dalbavancin against echovirus 1, ezetimibe against human immunodeficiency virus 1 and Zika virus, as well as azacitidine, cyclosporine, minocycline, oritavancin and ritonavir against Rift valley fever virus. Thus, the spectrum of antiviral activities of existing antiviral agents could be expanded towards other viral diseases.
Subject(s)
Antiviral Agents/pharmacology , DNA Viruses/drug effects , RNA Viruses/drug effects , Virus Diseases/drug therapy , Drug Repositioning , HumansABSTRACT
We studied retrospectively the outcome of Epstein-Barr virus (EBV)-related disease with EBV monitoring and preemptive rituximab to prevent post-transplant lymphoproliferative disorder (PTLD) in 319 consecutive allogeneic stem cell transplantations 2004-2012. Patients who received anti-thymocyte globulin (ATG) or alemtuzumab were regarded as high-risk for PTLD (n = 214). EBV DNAemia ≥1000 copies/mL plasma was observed in 50 (23%) of the high-risk patients. Thirty-three of the high-risk (15%) and one of the low-risk (1%) patients received rituximab, in combination with reduction of immunosuppression (n = 24) or chemotherapy (n = 4). Although rituximab was initiated only 5 d after first EBV load ≥1000 copies/mL, 85% of the rituximab-treated patients developed symptoms (lymphadenopathy 50%, fever 76%, and encephalitis/meningitis 12%). Response-rate to EBV treatment was 88%. Overall survival at 1- and 5-year was 71 and 52% for rituximab-treated patients, which was not inferior to all other patients post-transplant. In conclusion, rituximab therapy for EBV DNAemia does not affect long-term survival negatively.
Subject(s)
DNA, Viral/blood , Epstein-Barr Virus Infections/complications , Hematopoietic Stem Cell Transplantation/mortality , Lymphoproliferative Disorders/mortality , Rituximab/therapeutic use , Viremia/mortality , Adolescent , Antineoplastic Agents, Immunological/therapeutic use , Child , Child, Preschool , Combined Modality Therapy , DNA, Viral/genetics , Epstein-Barr Virus Infections/virology , Female , Follow-Up Studies , Herpesvirus 4, Human/isolation & purification , Humans , Infant , Lymphoproliferative Disorders/etiology , Lymphoproliferative Disorders/therapy , Male , Prognosis , Retrospective Studies , Risk Factors , Survival Rate , Transplantation, Homologous , Viral Load , Viremia/etiology , Viremia/therapyABSTRACT
BACKGROUND: Mycoplasma pneumoniae is a common cause of respiratory infections which can cause life-threatening pneumonia and serious extrapulmonary manifestations. Since the year 2000, the emergence of macrolide-resistant M. pneumoniae strains has increased with varying incidences across countries. In China more than 90% of the strains are resistant. M. pneumoniae diagnostics is mostly done with molecular methods, and in Sweden antibiotic resistance surveillance is not routinely performed. The prevalence of macrolide-resistant M. pneumoniae has not previously been studied in Sweden. MATERIAL AND METHODS: A total of 563 M. pneumoniae-positive respiratory samples, collected from four counties in Sweden between 1996 and 2013, were screened for mutations associated with macrolide resistance using a duplex FRET real-time PCR method. The real-time PCR targets the 23S rRNA gene, and differentiation between wild-type and resistant strains was achieved with a melting curve analysis. RESULTS: Of the 563 samples included, 548 were analyzed for mutations associated with macrolide resistance. No mutations were found. The detection rate of macrolide-resistant M. pneumoniae in this study was 0% [0.00-0.84%]. CONCLUSION: No macrolide-resistant M. pneumoniae has been detected in Sweden. However, the emergence and spread of macrolide-resistant M. pneumoniae strains in many countries commands continuous epidemiological surveillance.
ABSTRACT
Surgically removed palatine tonsils provide a conveniently accessible source of T and B lymphocytes to study the interplay between foreign pathogens and the host immune system. In this study we have characterised the distribution of human adenovirus (HAdV), Epstein-Barr virus (EBV) and human cytomegalovirus (HCMV) in purified tonsillar T and B cell-enriched fractions isolated from three patient age groups diagnosed with tonsillar hypertrophy and chronic/recurrent tonsillitis. HAdV DNA was detected in 93 out of 111 patients (84%), while EBV DNA was detected in 58 patients (52%). The most abundant adenovirus type was HAdV-5 (68%). None of the patients were positive for HCMV. Furthermore, 43 patients (39%) showed a co-infection of HAdV and EBV. The majority of young patients diagnosed with tonsillar hypertrophy were positive for HAdV, whereas all adult patients diagnosed with chronic/recurrent tonsillitis were positive for either HAdV or EBV. Most of the tonsils from patients diagnosed with either tonsillar hypertrophy or chronic/recurrent tonsillitis showed a higher HAdV DNA copy number in T compared to B cell-enriched fraction. Interestingly, in the majority of the tonsils from patients with chronic/recurrent tonsillitis HAdV DNA was detected in T cells only, whereas hypertrophic tonsils demonstrated HAdV DNA in both T and B cell-enriched fractions. In contrast, the majority of EBV positive tonsils revealed a preference for EBV DNA accumulation in the B cell-enriched fraction compared to T cell fraction irrespective of the patients' age.
Subject(s)
Adenovirus Infections, Human/diagnosis , Epstein-Barr Virus Infections/diagnosis , Lymphocytes/virology , Palatine Tonsil/virology , Tonsillitis/virology , Adenoviruses, Human/pathogenicity , Adolescent , Adult , Antigens, CD20/metabolism , Child , Child, Preschool , Cytomegalovirus/pathogenicity , Female , Genotype , Humans , Infant , Male , Phylogeny , Polymerase Chain Reaction , Young AdultABSTRACT
OBJECTIVE: It is assumed that cytomegaloviral (CMV) infection in inflammatory bowel disease (IBD) is caused by reactivation due to the immunosuppressive therapy, but the role of CMV as a pathophysiological factor and prognostic marker in IBD is unclear. The aim of this study was to investigate CMV infection in IBD, with real-time polymerase chain reaction (PCR) and immunohistochemistry, with emphasis on newly diagnosed disease. MATERIALS AND METHODS: In this prospective, controlled study, 67 patients with IBD and 34 control patients with irritable bowel syndrome (IBS) or rectal bleeding were included. Serology for CMV was analysed along with CMV DNA in plasma, mucosal biopsies, and faeces. Mucosal biopsies were further analysed with histopathology and CMV immunohistochemistry. RESULTS: Detection of CMV IgM was more common in patients with IBD, compared to controls, 21% versus 3%. CMV DNA was found in 16% of patients with newly diagnosed, untreated IBD and in 38% of steroid-treated patients. Four of the five patients that needed urgent surgery were CMV-DNA positive in at least one of three sample types. None of the controls had detectable CMV DNA. CONCLUSIONS: Active CMV infection was found in high proportions of newly diagnosed untreated patients with IBD, in patients on immunosuppression and in patients in the need of surgery. Low CMV-DNA levels in non-immunosuppressed patients were not a risk factor for the development of more severe IBD, while the detection of CMV DNA in patients on immunosuppressive therapy may foresee disease progression.
Subject(s)
Cytomegalovirus Infections/diagnosis , DNA, Viral/analysis , Immunosuppression Therapy/adverse effects , Inflammatory Bowel Diseases/complications , Inflammatory Bowel Diseases/drug therapy , Adolescent , Adult , Aged , Case-Control Studies , Cytomegalovirus , Feces/virology , Female , Humans , Immunohistochemistry , Male , Middle Aged , Prospective Studies , Real-Time Polymerase Chain Reaction , Risk Factors , Severity of Illness Index , Sweden , Young AdultABSTRACT
BACKGROUND: The future treatment of hepatitis C virus (HCV) infection will be combinations of direct-acting antivirals (DAAs) that not only target multiple viral targets, but are also effective against different HCV genotypes. Of the many drug targets in HCV, one promising target is the non-structural 5A protein (NS5A), against which inhibitors, namely daclatasvir, ledipasvir and ombitasvir, have shown potent efficacy. However, since HCV is known to have very high sequence diversity, development of resistance is a problem against but not limited to NS5A inhibitors (i.e. resistance also found against NS3-protease and NS5B non-nucleoside inhibitors), when used in suboptimal combinations. Furthermore, it has been shown that natural resistance against DAAs is present in treatment-naïve patients and such baseline resistance will potentially complicate future treatment strategies. METHODS: A pan-genotypic population-sequencing method with degenerated primers targeting the NS5A region was developed. We have investigated the prevalence of baseline resistant variants in 127 treatment-naïve patients of HCV genotypes 1a, 1b, 2b and 3a. RESULTS: The method could successfully sequence more than 95% of genotype 1a, 1b and 3a samples. Interpretation of fold resistance data against the NS5A inhibitors was done with the help of earlier published phenotypic data. Baseline resistance variants associated with high resistance (1000-50,000-fold) was found in three patients: Q30H or Y93N in genotype 1a patients and further Y93H in a genotype 3a patient. CONCLUSION: Using this method, baseline resistance can be examined and the data could have a potential role in selecting the optimal and cost-efficient treatment for the patient.
Subject(s)
Antiviral Agents/pharmacology , Hepacivirus/drug effects , Hepacivirus/genetics , Hepatitis C, Chronic/virology , Hepatitis C/virology , Polymorphism, Genetic , Viral Nonstructural Proteins/antagonists & inhibitors , Viral Nonstructural Proteins/genetics , Antiviral Agents/therapeutic use , Carbamates , Drug Resistance, Viral/genetics , Genotype , Imidazoles/therapeutic use , Mutation, Missense , Phylogeny , Polymerase Chain Reaction/methods , Prevalence , Protease Inhibitors/therapeutic use , Pyrrolidines , Sequence Analysis , Sweden/epidemiology , Valine/analogs & derivativesABSTRACT
The future interferon-free treatment of hepatitis C virus (HCV) infection could include NS3 protease inhibitors (PIs) for potent pan-genotypic effect. We studied the prevalence of pre-existing PI resistance associated amino acid variants (RAVs) in 126 treatment-naive patient samples of HCV genotypes 1a, 2b and 3a, the most common genotypes in Sweden. The NS3 genes were each amplified by nested PCR method with degenerated primers to enable a broad genotype analysis. Population sequencing method was used, and the sequences were aligned with the NS3 sequence from HCV genotype 1a H77 strain. Interpretation of fold-change resistance to NS3 candidate drugs were done from already published phenotypic resistance data. The prevalence of known PI RAVs at baseline in genotype 1a was 28% (15/53), either single (V36L or Q80K/R) or combinations (T54A/S and V55A/I) of mutation(s). In genotype 2b, specific mutations like V36L, Q80G and S122R of viral NS3 protease gene were found in 100% (11/11). These may be the natural polymorphisms unique to genotype 2b. Similarly, specific mutations like V36L and D168Q were found uniquely in all 3a samples (30/30). The natural PI RAVs found in genotype 1a, although with relatively weak resistance, could still render up to 10-fold-resistance to the approved (boceprevir and telaprevir) and the 2nd generation PIs (faldaprevir and simeprevir). Moreover, the natural polymorphisms in genotype 2b (i.e. S122R) and 3a (i.e. D168Q), with inherent PI drug resistance of up to 20 and 700 fold respectively, would explain why current PIs are primarily directed against genotype 1.
Subject(s)
Antiviral Agents/pharmacology , Drug Resistance, Viral , Hepacivirus/drug effects , Polymorphism, Genetic , Protease Inhibitors/pharmacology , Viral Nonstructural Proteins/antagonists & inhibitors , Viral Nonstructural Proteins/genetics , Amino Acid Substitution , Genotype , Hepacivirus/enzymology , Hepacivirus/genetics , Hepacivirus/isolation & purification , Hepatitis C, Chronic/virology , Humans , Mutation, Missense , SwedenABSTRACT
In contrast to ordinary PCRs, which have a limited multiplex capacity and often return false-negative results due to target variation or inhibition, our new detection strategy, VOCMA (variation-tolerant capture multiplex assay), allows variation-tolerant, target-specific capture and detection of many nucleic acids in one test. Here we demonstrate the use of a single-tube, dual-step amplification strategy that overcomes the usual limitations of PCR multiplexing, allowing at least a 22-plex format with retained sensitivity. Variation tolerance was achieved using long primers and probes designed to withstand variation at known sites and a judicious mix of degeneration and universal bases. We tested VOCMA in situations where enrichment from a large sample volume with high sensitivity and multiplexity is important (sepsis; streptococci, enterococci, and staphylococci, several enterobacteria, candida, and the most important antibiotic resistance genes) and where variation tolerance and high multiplexity is important (gastroenteritis; astrovirus, adenovirus, rotavirus, norovirus genogroups I and II, and sapovirus, as well as enteroviruses, which are not associated with gastroenteritis). Detection sensitivities of 10 to 1,000 copies per reaction were achieved for many targets. VOCMA is a highly multiplex, variation-tolerant, general purpose nucleic acid detection concept. It is a specific and sensitive method for simultaneous detection of nucleic acids from viruses, bacteria, fungi, and protozoa, as well as host nucleic acid, in the same test. It can be run on an ordinary PCR and a Luminex machine and is suitable for both clinical diagnoses and microbial surveillance.
Subject(s)
Clinical Laboratory Techniques/methods , Communicable Diseases/diagnosis , Molecular Diagnostic Techniques/methods , Nucleic Acid Amplification Techniques/methods , Polymorphism, Genetic , Animals , Bacteria/genetics , Bacteria/isolation & purification , Fungi/genetics , Fungi/isolation & purification , Humans , Parasites/genetics , Parasites/isolation & purification , Sensitivity and Specificity , Viruses/genetics , Viruses/isolation & purificationSubject(s)
Antiviral Agents/therapeutic use , Hepatitis C, Chronic/drug therapy , Protease Inhibitors/therapeutic use , Antiviral Agents/adverse effects , Drug Approval , Drug Resistance, Viral/genetics , Drug Therapy, Combination , Hepacivirus/genetics , Hepatitis C, Chronic/complications , Heterocyclic Compounds, 3-Ring/therapeutic use , Humans , Oligopeptides/therapeutic use , Proline/analogs & derivatives , Proline/therapeutic use , Protease Inhibitors/adverse effects , Simeprevir , Sulfonamides/therapeutic use , Treatment Outcome , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/geneticsABSTRACT
Genotyping of respiratory syncytial (RS) virus group A, by means of a novel method based on PCR, FRET (fluorescence resonance energy transmission) detection and two-dimensional melting curve analysis, was carried out on 80 RS virus samples of group A collected in Stockholm from 1976 to 2005. The Tm values were assessed for three different genotypes (GA2, GA5 and GA7) circulating in Sweden. Two pairs of probes were used and results of subsequent data analysis were plotted in a two-dimensional system. The results obtained were compared to genotyping using conventional nucleotide sequencing and phylogenetic tree analysis. It was found that the new assay was able to correctly identify genotype in about 89% of the isolates; it identified the remaining 11% as untypeable and as candidates for conventional nucleotide sequencing. The new method constitutes a complement to nucleotide sequencing and could be useful in studies of large numbers of samples in epidemiological studies.
Subject(s)
Molecular Epidemiology , Respiratory Syncytial Virus Infections/virology , Respiratory Syncytial Viruses/classification , Respiratory Tract Infections/virology , Reverse Transcriptase Polymerase Chain Reaction/methods , Adult , Child, Preschool , Fluorescence Resonance Energy Transfer , Humans , Infant , Phylogeny , Respiratory Syncytial Viruses/genetics , Sweden , Urban Population , Viral Fusion Proteins/geneticsABSTRACT
A real-time PCR was designed for detection of Chlamydophila pneumoniae and Mycoplasma pneumoniae such that each pathogen could be detected in a single tube and differentiated using molecular beacons marked with different fluorochromes. This duplex PCR, targeting the P1 adhesion gene for M. pneumoniae and the ompA gene for C. pneumoniae, was compared with two conventional PCR assays targeting the 16S rRNA gene and the ompA gene. A total of 120 clinical throat and nasopharyngeal swab samples were tested. DNA extraction was performed using an alkali denaturation/neutralization method, and real-time amplification, detection, and data analysis were performed using a Rotor-Gene 2000 real-time rotary analyzer (Corbett Life Science, Sydney, Australia). Using conventional PCR as a reference in an analysis of 120 samples, 13 of 14 samples positive for C. pneumoniae were detected by the novel real-time PCR. In an analysis of M. pneumoniae, 22 samples were positive in the conventional PCR and the novel assay detected 24 positive samples. When using the conventional PCR as a reference, sensitivity and specificity were 93% and 100%, respectively, for C. pneumoniae and 100% and 98%, respectively, for M. pneumoniae. With an overall agreement of 98.8%, this suggests that performance of the new duplex real-time PCR is comparable to that of conventional PCR.
Subject(s)
Chlamydophila Infections/diagnosis , Chlamydophila pneumoniae/isolation & purification , Mycoplasma pneumoniae/isolation & purification , Pneumonia, Mycoplasma/diagnosis , Polymerase Chain Reaction/methods , Adhesins, Bacterial/genetics , Bacterial Outer Membrane Proteins/genetics , DNA Primers/genetics , DNA, Bacterial/genetics , DNA, Bacterial/isolation & purification , Humans , Pharynx/microbiology , Sensitivity and SpecificityABSTRACT
A real-time PCR method targeting the Bordetella pertussis IS481 gene fragment was evaluated in a vaccine trial setting in which real-time PCR results could be validated against culture and serology results. Two commonly used DNA extraction methods, Amplicor Respiratory Preparation kit and the QIAamp DNA Mini Kit, were compared. An approximately 50-fold higher sensitivity was achieved using the Amplicor kit. 89 of 276 aspirates analysed with the IS481 real-time PCR were positive. Interestingly, six of these were culture negative and came from serology-negative patients. Defining true positive cases either as culture-positive or as PCR-positive cases that had been confirmed with a serology-positive result or verified with a newly constructed recA PCR, the sensitivity and specificity of the IS481 real-time PCR were 89% and 98%, respectively. This study confirms the specificity and high diagnostic sensitivity of IS481-based PCR methods for diagnosis of B. pertussis.
Subject(s)
Bordetella pertussis/isolation & purification , DNA, Bacterial/analysis , Pertussis Vaccine , Reverse Transcriptase Polymerase Chain Reaction/methods , Whooping Cough/diagnosis , Bordetella pertussis/genetics , Clinical Trials as Topic , DNA Primers , Humans , Sensitivity and Specificity , Whooping Cough/prevention & controlABSTRACT
The present study reports the diversity of rotavirus strains circulating in León, Nicaragua during three years. There was a shift of G and P genotypes with increment of one specific genotype during the second most important peak of diarrhea occurring in the beginning of every year.
Subject(s)
Rotavirus Infections/epidemiology , Rotavirus/classification , Rotavirus/genetics , Child, Preschool , Genotype , Humans , Nicaragua/epidemiology , Oligonucleotide Array Sequence Analysis , Prevalence , RNA, Viral/isolation & purification , Reverse Transcriptase Polymerase Chain Reaction , Rotavirus/metabolism , Rotavirus Infections/virology , Sequence Analysis, DNAABSTRACT
We describe two newly developed methods for rapid typing of the pertussis toxin subunit 1 gene (ptxS1). A real-time PCR assay based on hybridization probes and a Pyrosequencing assay were developed and the specificity, sensitivity, cost, hands-on time and post-assay data processing were compared to Sanger sequencing. Both methods enabled discrimination of all four allelic variants, correctly identified all ptxS1 alleles of 143 strains tested and proved suitable for large-scale screening of B. pertussis strains.
Subject(s)
Bordetella pertussis/genetics , Pertussis Toxin/genetics , Alleles , Bordetella pertussis/classification , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , Humans , Pertussis Toxin/chemistry , Pertussis Vaccine/genetics , Polymerase Chain Reaction , Polymorphism, Single Nucleotide , Sequence Analysis, DNA , Whooping Cough/microbiologyABSTRACT
A duplex quantitative real-time PCR (qPCR) assay was designed to detect both the polymerase gene (pol) and the glycoprotein gene (gB) of cytomegalovirus (CMV). The detection limit of the qPCR was determined to be 1 to 3 copies/reaction and the linear measure interval was 10(3) to 10(8) copies/ml. The qPCR system was compared to the COBAS Amplicor CMV Monitor test (COBAS) by an analysis of 138 plasma samples. Both systems detected CMV in 71 cases and had negative results for 33 samples. In addition, 34 samples were positive by qPCR and negative by the COBAS assay, but in no case was the COBAS result positive and the qPCR result negative. Thus, qPCR detected 48% more positive cases than the COBAS method. For samples with > or = 10(5) copies/ml by qPCR, a saturation effect was seen in the COBAS assay and quantification required dilution. Copy numbers for pol and gB by qPCR generally agreed. However, the reproducibility of qPCR assays and the need for an international standard are discussed. Discrepant copy numbers for pol and gB by qPCR were found for samples from two patients, and sequence analysis revealed that the corresponding CMV strains were mismatched at four nucleotide positions compared with the gB fragment primer sequences. In conclusion, a duplex qPCR assay in a real-time format facilitates quantitative measurements and minimizes the risk of false-negative results.