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PURPOSE: To report a rare presentation of a proven case of Mycobacterium chimaera infection presenting as multifocal choroiditis with recurrent choroidal neovascular membrane (CNVM) in one eye, initially misdiagnosed as punctate inner choroidopathy and later developed serpiginous-like choroiditis in the other eye. METHODS: Retrospective case report with a review of existing literature. RESULTS: A 30-year-old women presented with metamorphopsia (OD) and best-corrected visual acuity (BCVA) of 6/24 (OD) and was diagnosed to have punctate inner choroidopathy with CNVM (OD). Since then, she had received four intravitreal anti-vascular endothelial growth factor injections over 3 years. Two years later, she developed a slowly progressing choroidal lesion radiating from the disc in a serpiginoid manner in the left eye. There was no vitritis. Labs revealed a positive QuantiFERON-TB Gold test. High-resolution computed tomography of the thorax showed sub-centimetre noncalcified lymph nodes in subcarinal and perivascular regions, minimal pleural thickening in left lower zone, minimal pericardial effusion, bronchiectatic changes, and fibrotic strands in right middle and left lower lobes. Bronchoalveolar lavage grew M. chimaera intracellularae (matrix-assisted laser desorption/ionization time-of-flight mass spectrometry). She was given a course of clarithromycin, moxifloxacin, rifampicin, and doxycycline for 12 months. Though the right eye remained stable, choroidal lesion in the left eye continued to progress threatening the fovea, requiring oral steroids, methotrexate, and an intravitreal dexamethasone implant. At the last follow-up, her BCVA was 6/18 (OD) and 6/6 (OS). Both eyes were stable. CONCLUSION: This case highlights a rare presentation of proven M. chimaera infection presenting as multifocal choroiditis with recurrent CNVM in one eye and serpiginous-like choroiditis in the other eye, requiring aggressive treatment to salvage the vision.
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The continued emergence of vaccine-resistant SARS-CoV-2 variants of concern (VOC) requires specific identification of each VOC as it arises. Here, we report an expanded version of our previously described sloppy molecular beacon (SMB) melting temperature (Tm) signature-based assay for VOCs, now modified to include detection of Delta (B.1.617.2) and Omicron (B.1.1.529) sub-variants. The SMB-VOC assay targets the signature codons 501, 484 and 452 in the SARS-CoV-2 spike protein which we show can specifically detect and differentiate all known VOCs including the Omicron subvariants (BA.1, BA.2, BA.2.12.1, BA.4/BA.5). The limit of detection (LOD) of the assay was 20, 22 and 36 genomic equivalents (GE) per reaction with the Delta, Omicron BA.1 and BA.2 respectively. Clinical validation of the 3-codon assay in the LC480 instrument showed the assay detected 94% (81/86) of the specimens as WT or VOCs and 6% (5/86) of the tests producing indeterminate results compared to sequencing. Sanger sequencing also failed for four samples. None of the specimens were incorrectly identified as WT or as a different VOC by our assay. Thus, excluding specimens with indeterminant results, the assay was 100% sensitive and 100% specific compared to Sanger sequencing for variant identification. This new assay concept can be easily expanded to add newer variants and can serve as a robust diagnostic tool for selecting appropriate monoclonal antibody therapy and rapid VOC surveillance.
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COVID-19 , Magnoliopsida , Humanos , COVID-19/diagnóstico , Reação em Cadeia da Polimerase Via Transcriptase Reversa , SARS-CoV-2/genética , Temperatura , Teste para COVID-19RESUMO
BACKGROUND: Upper respiratory samples used to test for SARS-CoV-2 virus may be infectious and present a hazard during transport and testing. A buffer with the ability to inactivate SARS-CoV-2 at the time of sample collection could simplify and expand testing for COVID-19 to non-conventional settings. METHODS: We evaluated a guanidium thiocyanate-based buffer, eNAT™ (Copan) as a possible transport and inactivation medium for downstream Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) testing to detect SARS-CoV-2. Inactivation of SARS-CoV-2 USA-WA1/2020 in eNAT and in diluted saliva was studied at different incubation times. The stability of viral RNA in eNAT was also evaluated for up to 7 days at room temperature (28°C), refrigerated conditions (4°C) and at 35°C. RESULTS: SARS-COV-2 virus spiked directly in eNAT could be inactivated at >5.6 log10 PFU/ml within a minute of incubation. When saliva was diluted 1:1 in eNAT, no cytopathic effect (CPE) on VeroE6 cells was observed, although SARS-CoV-2 RNA could be detected even after 30 min incubation and after two cell culture passages. A 1:2 (saliva:eNAT) dilution abrogated both CPE and detectable viral RNA after as little as 5 min incubation in eNAT. SARS-CoV-2 RNA from virus spiked at 5X the limit of detection remained positive up to 7 days of incubation in all tested conditions. CONCLUSION: eNAT and similar guanidinium thiocyanate-based media may be of value for transport, stabilization, and processing of clinical samples for RT-PCR based SARS-CoV-2 detection.
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Teste de Ácido Nucleico para COVID-19/métodos , COVID-19/diagnóstico , Guanidina/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/genética , Saliva/efeitos dos fármacos , Saliva/virologia , Manejo de Espécimes/métodos , Inativação de Vírus/efeitos dos fármacos , Animais , COVID-19/virologia , Chlorocebus aethiops , Meios de Cultura , Voluntários Saudáveis , Humanos , RNA Viral/genética , Células VeroRESUMO
Nucleic acid amplification tests (NAATs) are the primary means of identifying acute infections caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Accurate and fast test results may permit more efficient use of protective and isolation resources and allow rapid therapeutic interventions. We evaluated the analytical and clinical performance characteristics of the Xpert Xpress SARS-CoV-2 (Xpert) test, a rapid, automated molecular test for SARS-CoV-2. Analytical sensitivity and specificity/interference were assessed with infectious SARS-CoV-2; other infectious coronavirus species, including SARS-CoV; and 85 nasopharyngeal swab specimens positive for other respiratory viruses, including endemic human coronaviruses (hCoVs). Clinical performance was assessed using 483 remnant upper- and lower-respiratory-tract specimens previously analyzed by standard-of-care (SOC) NAATs. The limit of detection of the Xpert test was 0.01 PFU/ml. Other hCoVs, including Middle East respiratory syndrome coronavirus, were not detected by the Xpert test. SARS-CoV, a closely related species in the subgenus Sarbecovirus, was detected by a broad-range target (E) but was distinguished from SARS-CoV-2 (SARS-CoV-2-specific N2 target). Compared to SOC NAATs, the positive agreement of the Xpert test was 219/220 (99.5%), and the negative agreement was 250/261 (95.8%). A third tie-breaker NAAT resolved all but three of the discordant results in favor the Xpert test. The Xpert test provided sensitive and accurate detection of SARS-CoV-2 in a variety of upper- and lower-respiratory-tract specimens. The high sensitivity and short time to results of approximately 45 min may impact patient management.
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Betacoronavirus/isolamento & purificação , Técnicas de Laboratório Clínico/métodos , Infecções por Coronavirus/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Pneumonia Viral/diagnóstico , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Automação Laboratorial/métodos , Betacoronavirus/genética , COVID-19 , Teste para COVID-19 , Criança , Pré-Escolar , Infecções por Coronavirus/virologia , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Nasofaringe/virologia , Pandemias , Pneumonia Viral/virologia , SARS-CoV-2 , Sensibilidade e Especificidade , Adulto JovemRESUMO
Francisella tularensis, Bacillus anthracis, and Yersinia pestis are tier 1 select agents with the potential to rapidly cause severe disease. Rapid detection of these bacteria from patient samples at the point of care could contribute to improved clinical outcomes in the event of a bioterrorism attack. A multiplex nested PCR assay for detection of F. tularensis, B. anthracis, and Y. pestis directly from patient blood samples was developed using the GeneXpert system. The multiplex GeneXpert cartridge-based assay includes all necessary sample processing and amplification reagents. Blood samples spiked with different numbers of CFU were used to measure the analytical limit of detection (LOD) and dynamic range. Sensitivity was determined by testing spiked blood samples and negative-control blood in a blind manner. Specificity was determined by testing against nontarget pathogens and blood samples from clinical patients. The assay LOD was 8.5 CFU/ml for F. tularensis, 10 CFU/ml for B. anthracis, and 4.5 CFU/ml for Y. pestis The sensitivity was 100% at the LOD for all three select agent bacteria in spiked patient blood samples. The assay specificity was 100% when it was tested against both nontarget pathogens and clinical patient blood samples. The total assay time was approximately 100 min. This automated assay, which is suitable for use at the point of care, identifies three select agents directly in blood without the need for enrichment with a high sensitivity within 100 min. This assay may enable rapid detection and treatment of patients infected with the target organisms in the event of a bioterrorism attack.
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Bacillus anthracis/isolamento & purificação , Sangue/microbiologia , Francisella tularensis/isolamento & purificação , Reação em Cadeia da Polimerase Multiplex , Yersinia pestis/isolamento & purificação , Antraz/sangue , Antraz/diagnóstico , Ensaios de Triagem em Larga Escala , Humanos , Limite de Detecção , Peste/sangue , Peste/diagnóstico , Sensibilidade e Especificidade , Tularemia/sangue , Tularemia/diagnósticoRESUMO
Background: Influenza viruses have emerged as virulent pathogens causing considerable burden across the world. A thorough understanding of the pattern in occurrence of influenza globally is the need of hour. The present study deals with analysis of the dynamics of Influenza virus, especially the influence of seasonal change on viral circulation and causation of epidemics/pandemics in the context of subtropical region. Methods: During the 7 year (2009-2015) study, 36670 specimens were subjected to influenza analysis. Nasopharyngeal swabs collected from suspected patients from Chennai, Tamil Nadu, were tested and typed by real-time polymerase chain reaction assay. Results: During 2009 pandemic, among influenza A positives 95.16% were Apdm09, indicating that there was a predominant circulation of Apdm09. During postpandemic period, there were waves in the occurrence of Apdm09 which indicates fall in immunity with buildup in the susceptible population. Conclusion: In Chennai, Tamil Nadu, influenza positivity started with the onset of monsoon and peaks during the postmonsoon months throughout the study period. The assessment of meteorological factors compounding influenza activity can help in raising alerts to the public health officials of impending disaster which suggests that Influenza vaccination can be initiated before monsoon months in South India.
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Vírus da Influenza A/patogenicidade , Influenza Humana/virologia , Humanos , Índia , Vírus da Influenza A/genética , Influenza Humana/genética , Reação em Cadeia da Polimerase em Tempo Real , Estações do Ano , Síndrome Respiratória Aguda Grave/virologiaRESUMO
Dengue fever is a severe, widespread disease with more than 2 million diagnosed infections per year. The Dengue virus protease represents a cardinal target for prudent drug design. Among the four serotypes Dengue 2 is known for the occurrence of its frequent epidemics. The new compound inhibited the Dengue-2 in the low-micromolar range in cells. At the moment, protease inhibitors are not actively tried against dengue virus as therapeutic option. We have identified thiosemicarbazones derived phenyl-acetyl ketones as candidate for a novel class of protease inhibitors. Here, we report the selective and non-competitive inhibition of the Dengue virus serotype 2 in vitro and in silico. Molecular docking suggests binding at a specific active site. In addition to the docking assays, few techniques were developed to interpret these molecules's antiviral profile in vitro.
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Antivirais/farmacologia , Vírus da Dengue/efeitos dos fármacos , Inibidores de Proteases/farmacologia , Serina Endopeptidases , Animais , Domínio Catalítico , Chlorocebus aethiops , Descoberta de Drogas , Simulação de Acoplamento Molecular , Células VeroRESUMO
Skin cancer is the most common form of cancer and is globally rising. Historically, the diagnosis of skin cancers has depended on various conventional techniques which are of an invasive manner. A variety of commercial diagnostic tools and auxiliary techniques are available to detect skin cancer. This article explains in detail the principles and approaches involved for non-invasive skin cancer diagnostic methods such as photography, dermoscopy, sonography, confocal microscopy, Raman spectroscopy, fluorescence spectroscopy, terahertz spectroscopy, optical coherence tomography, the multispectral imaging technique, thermography, electrical bio-impedance, tape stripping and computer-aided analysis. The characteristics of an ideal screening test are outlined, and the authors pose several points for clinicians and scientists to consider in the evaluation of current and future studies of skin cancer detection and diagnosis. This comprehensive review critically analyses the literature associated with the field and summarises the recent updates along with their merits and demerits.
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The Xpert MTB/RIF assay (Xpert) is a rapid test for tuberculosis (TB) and rifampin resistance (RIF-R) suitable for point-of-care testing. However, it has decreased sensitivity in smear-negative sputum, and false identification of RIF-R occasionally occurs. We developed the Xpert MTB/RIF Ultra assay (Ultra) to improve performance. Ultra and Xpert limits of detection (LOD), dynamic ranges, and RIF-R rpoB mutation detection were tested on Mycobacterium tuberculosis DNA or sputum samples spiked with known numbers of M. tuberculosis H37Rv or Mycobacterium bovis BCG CFU. Frozen and prospectively collected clinical samples from patients suspected of having TB, with and without culture-confirmed TB, were also tested. For M. tuberculosis H37Rv, the LOD was 15.6 CFU/ml of sputum for Ultra versus 112.6 CFU/ml of sputum for Xpert, and for M. bovis BCG, it was 143.4 CFU/ml of sputum for Ultra versus 344 CFU/ml of sputum for Xpert. Ultra resulted in no false-positive RIF-R specimens, while Xpert resulted in two false-positive RIF-R specimens. All RIF-R-associated M. tuberculosis rpoB mutations tested were identified by Ultra. Testing on clinical sputum samples, Ultra versus Xpert, resulted in an overall sensitivity of 87.5% (95% confidence interval [CI], 82.1, 91.7) versus 81.0% (95% CI, 74.9, 86.2) and a sensitivity on sputum smear-negative samples of 78.9% (95% CI, 70.0, 86.1) versus 66.1% (95% CI, 56.4, 74.9). Both tests had a specificity of 98.7% (95% CI, 93.0, 100), and both had comparable accuracies for detection of RIF-R in these samples. Ultra should significantly improve TB detection, especially in patients with paucibacillary disease, and may provide more-reliable RIF-R detection.IMPORTANCE The Xpert MTB/RIF assay (Xpert), the first point-of-care assay for tuberculosis (TB), was endorsed by the World Health Organization in December 2010. Since then, 23 million Xpert tests have been procured in 130 countries. Although Xpert showed high overall sensitivity and specificity with pulmonary samples, its sensitivity has been lower with smear-negative pulmonary samples and extrapulmonary samples. In addition, the prediction of rifampin resistance (RIF-R) in paucibacillary samples and for a few rpoB mutations has resulted in both false-positive and false-negative results. The present study is the first demonstration of the design features and operational characteristics of an improved Xpert Ultra assay. This study also shows that the Ultra format overcomes many of the known shortcomings of Xpert. The new assay should significantly improve TB detection, especially in patients with paucibacillary disease, and provide more-reliable detection of RIF-R.
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Antibióticos Antituberculose/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/isolamento & purificação , Testes Imediatos , Rifampina/farmacologia , Tuberculose/diagnóstico , Proteínas de Bactérias/genética , RNA Polimerases Dirigidas por DNA/genética , Humanos , Limite de Detecção , Técnicas de Diagnóstico Molecular/métodos , Mutação , Mycobacterium tuberculosis/genética , Reação em Cadeia da Polimerase/métodos , Sensibilidade e Especificidade , Escarro/microbiologia , Tuberculose/microbiologia , Tuberculose Pulmonar/diagnóstico , Tuberculose Pulmonar/microbiologiaRESUMO
Bacillus anthracis is a tier 1 select agent with the potential to quickly cause severe disease. Rapid identification of this pathogen may accelerate treatment and reduce mortality in the event of a bioterrorism attack. We developed a rapid and sensitive assay to detect B. anthracis bacteremia using a system that is suitable for point-of-care testing. A filter-based cartridge that included both sample processing and PCR amplification functions was loaded with all reagents needed for sample processing and multiplex nested PCR. The assay limit of detection (LOD) and dynamic range were determined by spiking B. anthracis DNA into individual PCR mixtures and B. anthracis CFU into human blood. One-milliliter blood samples were added to the filter-based detection cartridge and tested for B. anthracis on a GeneXpert instrument. Assay specificity was determined by testing blood spiked with non-anthrax bacterial isolates or by testing blood samples drawn from patients with concurrent non-B. anthracis bacteremia or nonbacteremic controls. The assay LODs were 5 genome equivalents per reaction and 10 CFU/ml blood for both the B. anthracis Sterne and V1B strains. There was a 6-log10 dynamic range. Assay specificity was 100% for tests of non-B. anthracis bacterial isolates and patient blood samples. Assay time was less than 90 min. This automated system suitable for point-of-care detection rapidly identifies B. anthracis directly from blood with high sensitivity. This assay might lead to early detection and more rapid therapy in the event of a bioterrorism attack.
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Antraz/diagnóstico , Bacillus anthracis/genética , Bacteriemia/diagnóstico , DNA Bacteriano/sangue , Testes Imediatos , Bacillus anthracis/isolamento & purificação , Bacteriemia/microbiologia , DNA Bacteriano/genética , Sistemas Inteligentes , Genoma Bacteriano/genética , Humanos , Limite de DetecçãoRESUMO
Francisella tularensis is a potential bioterrorism agent that is highly infectious at very low doses. Diagnosis of tularemia by blood culture and nucleic acid-based diagnostic tests is insufficiently sensitive. Here, we demonstrate a highly sensitive F. tularensis assay that incorporates sample processing and detection into a single cartridge suitable for point-of-care detection. The assay limit of detection (LOD) and dynamic range were determined in a filter-based cartridge run on the GeneXpert system. F. tularensis DNA in buffer or CFU of F. tularensis was spiked into human or macaque blood. To simulate detection in human disease, the assay was tested on blood drawn from macaques infected with F. tularensis Schu S4 at daily intervals. Assay detection was compared to that with a conventional quantitative PCR (qPCR) assay and blood culture. The assay LOD was 0.1 genome equivalents (GE) per reaction and 10 CFU/ml F. tularensis in both human and macaque blood. In infected macaques, the assay detected F. tularensis on days 1 to 4 postinfection in 21%, 17%, 60%, and 83% of macaques, respectively, compared to conventional qPCR positivity rates of 0%, 0%, 30%, and 100% and CFU detection of blood culture at 0%, 0%, 0%, and 10% positive, respectively. Assay specificity was 100%. The new cartridge-based assay can rapidly detect F. tularensis in bloodstream infections directly in whole blood at the early stages of infection with a sensitivity that is superior to that of other methods. The simplicity of the automated testing procedures may make this test suitable for rapid point-of-care detection.
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Automação Laboratorial/métodos , Técnicas Bacteriológicas/métodos , Sangue/microbiologia , Francisella tularensis/isolamento & purificação , Técnicas de Diagnóstico Molecular/métodos , Tularemia/diagnóstico , Animais , Francisella tularensis/genética , Humanos , Macaca , Sensibilidade e EspecificidadeRESUMO
Dengue is a public health problem with an increasing global incidence and geographic distribution in almost all tropical and subtropical countries, with a transition from epidemic to endemic occurrence. In this study, we report a six-year analysis (2009-2014) performed at the Department of Virology, King Institute of Preventive Medicine, Chennai, Tamil Nadu, India. Our data confirm earlier findings that dengue is highly endemic in Chennai. In the present study, 10,099 serum samples from suspected dengue cases were tested for IgM ELISA (NIV Capture) and IgG Panbio ELISA (Australia). Of these suspected cases 6,798 and 3,301 were pediatric and adult cases, respectively, and 1,927 (19.08 %) were confirmed serologically as dengue. Of these, 1,752 (25.7 %) and 175 (5.3 %) were pediatric and adult cases, respectively. The aim of this study was to highlight the occurrence of DHF and DSS, mainly among the pediatric population, in which the infection causes higher mortality and morbidity. The overall positivity was higher in the pediatric group than in the adults. Detection of both IgM and IgG positivity will be useful for monitoring infection rates, the disease spectrum, and the prevalence of the different serotypes, which will give us insight about the circulating serotypes and pathogenicity. These data will be valuable for providing an early warning to predict an impending epidemic leading to major clinical manifestations of DHF and DSS.
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Dengue/epidemiologia , Dengue/patologia , Doenças Endêmicas , Adolescente , Adulto , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Anticorpos Antivirais/sangue , Criança , Pré-Escolar , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Índia/epidemiologia , Lactente , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Estudos Soroepidemiológicos , Adulto JovemRESUMO
BACKGROUND: Tuberculosis (TB) is difficult to diagnose in children using molecular tests, because children have difficulty providing respiratory samples. Stool could replace sputum for diagnostic TB testing if adequate sample processing techniques were available. METHODS: We developed a rapid method to process large volumes of stool for downstream testing by the Xpert MTB/RIF (Xpert) TB-detection assay. The method was tested and optimized on stool samples spiked with known numbers of M. tuberculosis colony forming units (CFU), and stools from M. tuberculosis-infected cynomolgus macaques (Macaca fascicularis). Performance was scored on number of positive Xpert tests, the cycle thresholds (Cts) of the Xpert sample-processing control (SPC), and the Cts of the M. tuberculosis-specific rpoB probes. The method was then validated on 20 confirmed TB cases and 20 controls in Durban, South Africa. RESULTS: The assay's analytical limit of detection was 1,000 CFU/g of stool. As much as one gram of spiked stool could be tested without showing increased PCR inhibition. In analytical spiking experiments using human stool, 1g samples provided the best sensitivity compared to smaller amounts of sample. However, in Macaques with TB, 0.6g stool samples performed better than either 0.2g or 1.2g samples. Testing the stool of pediatric TB suspects and controls suggested an assay sensitivity of 85% (95% CI 0.6-0.9) and 84% (95% CI 0.6-0.96) for 0.6g and 1.2g stool samples, respectively, and a specificity of 100% (95% CI 0.77-1) and 94% (95% CI 0.7-0.99), respectively. CONCLUSION: This novel approach may permit simple and rapid detection of TB using pediatric stool samples.
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Fezes/microbiologia , Mycobacterium tuberculosis/isolamento & purificação , Tuberculose/diagnóstico , Animais , Criança , Humanos , Macaca fascicularis , Macaca mulatta , Sensibilidade e Especificidade , Manejo de EspécimesRESUMO
The medicated wound dressing material with highly interconnected pores, mimicking the function of the extracellular matrix was fabricated for the promotion of cell growth. In this study, keratin (K), fibrin (F) and gelatin (G) composite scaffold (KFG-SPG) was fabricated by freeze drying technique and the mupirocin (D) drug was successfully incorporated with KFG-SPG (KFG-SPG-D) intended for tissue engineering applications. The fabrication of scaffold was performed without the use of any strong chemical solvents, and the solid sponge scaffold was obtained with well interconnected pores. The porous morphology of the scaffold was confirmed by SEM analysis and exhibited competent mechanical properties. KFG-SPG and KFG-SPG-D possess high level of biocompatibility, cell proliferation and cell adhesion of NIH 3T3 fibroblast and human keratinocytes (HaCaT) cell lines thereby indicating the scaffolds potential as a suitable medicated dressing for wound healing.
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Materiais Biomiméticos/química , Materiais Biomiméticos/farmacologia , Fibrina/química , Gelatina/química , Queratinas/química , Engenharia Tecidual/métodos , Alicerces Teciduais/química , Animais , Antibacterianos/química , Antibacterianos/farmacologia , Materiais Biocompatíveis/química , Materiais Biocompatíveis/farmacologia , Bovinos , Adesão Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Liberação Controlada de Fármacos , Estabilidade Enzimática/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Liofilização , Humanos , Camundongos , Células NIH 3T3 , Porosidade , Staphylococcus aureus/efeitos dos fármacos , Resistência à Tração , Cicatrização/efeitos dos fármacosRESUMO
BACKGROUND & OBJECTIVES: Human parainfluenza virus (HPIV) accounts for a significant proportion of lower respiratory tract infections in children as well as adults. This study was done to detect the presence of different subtypes of HPIV from patients having influenza like illness (ILI). METHODS: Throat and nasal swabs from 232 patients with ILI who were negative for influenza viruses were tested by multiplex reverse transcription polymerase chain reaction(mRT-PCR) for the detection of human parainfluenza virus. All samples were inoculated in rhesus monkey kidney (LLC-MK2) cell line. RESULTS: Of the 232 samples, 26(11.2%) were positive by mRT-PCR and nine (34.6%) showed cytopathic effect with syncytium formation for HPIV and all were HPIV-3 serotype, other serotypes like 1,2,4 were negative. The HPIV-3 strains (HN gene) were sequenced and analysed. Two novel mutations were identified at amino acid residues 295 and 297. INTERPRETATION & CONCLUSIONS: The mRT-PCR assay offers a rapid, sensitive and accurate diagnostic method for detection of HPIV which enables early detection and control. In our study there was a predominance of HPIV among 1-5 yr age group and the school going age group was less affected. Further studies need to be done to characterize HPIV isolated from different parts of the country.
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Vírus da Parainfluenza 1 Humana/isolamento & purificação , Vírus da Parainfluenza 2 Humana/isolamento & purificação , Infecções por Paramyxoviridae/virologia , Humanos , Índia , Vírus da Parainfluenza 1 Humana/genética , Vírus da Parainfluenza 2 Humana/genética , Infecções por Paramyxoviridae/fisiopatologiaRESUMO
The utility of the GeneXpert MTB/RIF (Xpert) assay for detection of Mycobacterium tuberculosis in sputum samples has been extensively studied. However, the performance of the Xpert assay as applied to other readily accessible body fluids such as exhaled breath condensate (EBC), saliva, urine, and blood has not been established. We used the Xpert assay to test EBC, saliva, urine, and blood samples from HIV-negative, smear- and culture-positive pulmonary tuberculosis (TB) patients for the presence of M. tuberculosis. To compare the ability of the assay to perform bacterial load measurements on sputum samples with versus without sample processing, the assay was also performed on paired direct and processed sputum samples from each patient. The Xpert assay detected M. tuberculosis in none of the 26 EBC samples (sensitivity, 0.0%; 95% confidence interval [95% CI], 0.0%, 12.9%), 10 of the 26 saliva samples (sensitivity, 38.5%; 95% CI, 22.4%, 57.5%), 1 of 26 urine samples (sensitivity, 3.8%; 95% CI, 0.7%, 18.9%), and 2 of 24 blood samples (sensitivity, 8.3%; 95% CI, 2.3%, 25.8%). For bacterial load measurements in the different types of sputum samples, the cycle thresholds of the two M. tuberculosis-positive sputum types were well correlated (Spearman correlation of 0.834). This study demonstrates that the Xpert assay should not be routinely used to detect M. tuberculosis in EBC, saliva, urine, or blood samples from HIV-negative patients suspected of having pulmonary tuberculosis. As a test of bacterial load, the assay produced similar results when used to test direct versus processed sputum samples. Sputum remains the optimal sample type for diagnosing pulmonary tuberculosis in HIV-negative patients with the Xpert assay.
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Técnicas Bacteriológicas/métodos , Líquidos Corporais/microbiologia , Técnicas de Diagnóstico Molecular/métodos , Mycobacterium tuberculosis/isolamento & purificação , Tuberculose Pulmonar/diagnóstico , Tuberculose Pulmonar/microbiologia , Adulto , Humanos , Mycobacterium tuberculosis/genética , Sensibilidade e EspecificidadeRESUMO
Tuberculosis (TB) remains a leading cause of death among HIV-infected adults, in part because of delayed diagnosis and therefore delayed initiation of treatment. Recently, the Gene-Xpert platform, a rapid, PCR-based diagnostic platform, has been validated for the diagnosis of TB with sputum. We have evaluated the Xpert MTB/RIF assay for the diagnosis of Mycobacterium tuberculosis bacteremia and investigated its impact on clinical outcomes. Consecutive HIV-infected adults with fever and cough presenting to Queen Elizabeth Central Hospital, Blantyre, Malawi, were recruited and followed up for 2 months. At presentation, three sputum samples were examined by smear, culture, and Xpert MTB/RIF assay for the presence of M. tuberculosis and blood was drawn for PCR with Xpert, for mycobacterial culture (Myco/F Lytic), and for aerobic culture. One hundred four patients were recruited, and 44 (43%) were sputum culture positive for M. tuberculosis. Ten were Xpert blood positive, for a sensitivity of 21% and a specificity of 100%. The 2-week mortality rate was significantly higher among patients who were Xpert blood positive than among those who were negative (40% versus 3%; multivariate odds ratio [OR] for death if positive, 44; 95% confidence interval [CI], 3 to 662). This effect persisted on assessment of the mortality rate at 2 months (40% versus 11%; OR, 5.6; 95% CI, 1.3 to 24.6). When screening uncomplicated patients presenting with a productive cough for pulmonary TB, Xpert blood offers no diagnostic advantage over sputum testing. Despite this, Xpert blood positivity is highly predictive of early death and this test rapidly identifies a group of patients in urgent need of initiation of treatment.
Assuntos
Bacteriemia/diagnóstico , Técnicas Bacteriológicas/métodos , Infecções por HIV/complicações , Técnicas de Diagnóstico Molecular/métodos , Mycobacterium tuberculosis/isolamento & purificação , Tuberculose/diagnóstico , Adulto , Estudos de Coortes , Feminino , Humanos , Malaui , Masculino , Pessoa de Meia-Idade , Mycobacterium tuberculosis/genética , Valor Preditivo dos Testes , Prognóstico , Estudos Prospectivos , Sensibilidade e Especificidade , Análise de SobrevidaRESUMO
We have developed a novel blood lysis-centrifugation approach for highly sensitive Mycobacterium tuberculosis detection in large volumes of blood with the Xpert MTB/RIF assay. One through 20 ml of blood was spiked with 0.25 to 10 CFU/ml of the M. tuberculosis surrogate M. bovis BCG. Multiple replicates of each sample were processed by a new lysis-centrifugation method and tested with the Xpert MTB/RIF assay. The assay was very sensitive with increased blood volumes. In the 20-ml samples, BCG was detected in blood spiked with 10, 5, 1, and 0.25 CFU/ml 100, 100, 83, and 57% of the time, respectively, compared to 100, 66, 18, and 18%, of the time, respectively, in 1-ml blood samples. Assay sensitivity was influenced by the type of anticoagulant used, with acid-citrate-dextrose solution B (ACD-B) providing the best results. A limit of detection of 10 CFU/ml was established with BCG spiked into ACD-B-treated blood, and 92, 36, and 33% of the samples with 5, 1, and 0.5 CFU/ml, respectively, were assay positive. The lysis buffer was stable both at room temperature and at 4°C for 2 months. The assay was tested with blood stored for 8 days without a change in sensitivity as measured by cycle threshold. This new assay format extends the capability of the Xpert MTB/RIF test, enabling up to 20 ml of blood to be tested rapidly for the presence of M. tuberculosis. This approach may be a useful method to detect extrapulmonary tuberculosis and the risk of death in immunocompromised patients.
Assuntos
Bacteriemia/diagnóstico , Técnicas Bacteriológicas/métodos , Técnicas de Diagnóstico Molecular/métodos , Mycobacterium tuberculosis/isolamento & purificação , Tuberculose/diagnóstico , Centrifugação/métodos , Humanos , Mycobacterium tuberculosis/genética , Sensibilidade e Especificidade , Manejo de Espécimes/métodosRESUMO
The three most common pathogenic species of Vibrio, Vibrio cholerae, Vibrio parahaemolyticus and Vibrio vulnificus, are of major concerns due to increased incidence of water- and seafood-related outbreaks and illness worldwide. Current methods are lengthy and require biochemical and molecular confirmation. A novel label-free forward light-scattering sensor was developed to detect and identify colonies of these three pathogens in real time in the presence of other vibrios in food or water samples. Vibrio colonies grown on agar plates were illuminated by a 635 nm laser beam and scatter-image signatures were acquired using a CCD (charge-coupled device) camera in an automated BARDOT (BActerial Rapid Detection using Optical light-scattering Technology) system. Although a limited number of Vibrio species was tested, each produced a unique light-scattering signature that is consistent from colony to colony. Subsequently a pattern recognition system analysing the collected light-scatter information provided classification in 1-2 min with an accuracy of 99%. The light-scattering signatures were unaffected by subjecting the bacteria to physiological stressors: osmotic imbalance, acid, heat and recovery from a viable but non-culturable state. Furthermore, employing a standard sample enrichment in alkaline peptone water for 6 h followed by plating on selective thiosulphate citrate bile salts sucrose agar at 30°C for â¼ 12 h, the light-scattering sensor successfully detected V. cholerae, V. parahaemolyticus and V. vulnificus present in oyster or water samples in 18 h even in the presence of other vibrios or other bacteria, indicating the suitability of the sensor as a powerful screening tool for pathogens on agar plates.