Characterization and prediction of viral loads of Hepatitis B serum samples by using surface-enhanced Raman spectroscopy (SERS).

BACKGROUND: Raman spectroscopy is a promising technique to analyze the body fluids for the purpose of non-invasive disease diagnosis. OBJECTIVES: To develop a surface-enhanced Raman spectroscopy (SERS) based method for qualitative and quantitative analysis of hepatitis B viral (HBV) infection from blood serum samples. METHODS: Clinically diagnosed hepatitis B virus (HBV) infected serum samples of patients of different levels of viral loads have been subjected for SERS analysis in comparison with the healthy ones by using silver nanoparticles (Ag NPs) based SERS substrates. The SERS measurements were performed on blood serum samples of 11 healthy and 32 clinically diagnosed HBV patients of different viral load levels of different exponentials including (101, 102 called as low level), (103, 104 called as medium level) and (105, 108 called as high level). Furthermore, multivariate data analysis techniques, Principal Component Analysis (PCA) and Partial Least Square Regression (PLSR) were also performed on SERS spectral data. RESULTS: The SERS spectral features due to biochemical changes in HBV positive serum samples associated with the increasing viral loads were established which could be employed for HBV diagnostic purpose. PCA was found helpful for the differentiation between SERS spectral data of serum samples of different levels of HBV infection and healthy individuals. PLSR model developed with standard samples of known viral loads for predicting the viral loads of blind/unknown samples with 99% predicted accuracy. CONCLUSION: SERS can be employed for qualitative and quantitative analysis of HBV infection from blood serum samples.

Surface-enhanced Raman spectroscopy for identification of food processing bacteria.

Food processing bacteria play important role in providing flavors, ingredients and other beneficial characteristics to the food but at the same time some bacteria are responsible for food spoilage. Therefore, quick and reliable identification of these food processing bacteria is very necessary for the differentiation between different species which may help in the development of more useful food processing methodologies. In this study, analysis of different bacterial species (Lactobacillus fermentum, Fructobacillus fructosus, Pediococcus pentosaceus and Halalkalicoccus jeotgali) was performed with our in-house developed Ag NPs-based surface-enhanced Raman spectroscopy (SERS) method. The SERS spectral data was analyzed by multivariate data analysis techniques including principal component analysis (PCA) and partial least square discriminant analysis (PLS-DA). Bacterial species were differentiated on the basis of SERS spectral features and potential of SERS was compared with the Raman spectroscopy (RS). SERS along with PCA and PLS-DA was found to be an efficient technique for identification and differentiation of food processing bacterial species. Differentiation with accuracy of 99.5% and sensitivity of 99.7% was depicted by PLS-DA model using leave one out cross validation.

Surface-enhanced Raman spectroscopy for the identification of tigecycline-resistant E. coli strains.

Tigecycline (TGC) is recognised as last resort of drugs against several antibiotic-resistant bacteria. Bacterial resistance to tigecycline due to presence of plasmid-mediated mobile TGC resistance genes (tet X3/X4) has broken another defense line. Therefore, rapid and reproducible detection of tigecycline-resistant E. coli (TREC) is required. The current study is designed for the identification and differentiation of TREC from tigecycline-sensitive E. coli (TSEC) by employing SERS by using Ag NPs as a SERS substrate. The SERS spectral fingerprints of E. coli strains associated directly or indirectly with the development of resistance against tigecycline have been distinguished by comparing SERS spectral data of TSEC strains with each TREC strain. Moreover, the statistical analysis including Principal Component Analysis (PCA), Hierarchical Cluster Analysis (HCA) and Partial Least Squares-Discriminant Analysis (PLS-DA) were employed to check the diagnostic potential of SERS for the differentiation among TREC and TSEC strains. The qualitative identification and differentiation between resistant and sensitive strains and among individual strains have been efficiently done by performing both PCA and HCA. The successful discrimination among TREC and TSEC at the strain level is performed by PLS-DA with 98% area under ROC curve, 100% sensitivity, 98.7% specificity and 100% accuracy.

Surface-enhanced Raman spectroscopy analysis of serum samples of typhoid patients of different stages.

BACKGROUND: Surface-enhanced Raman spectroscopy (SERS) of body fluids is considered a quick, simple and easy to use method for the diagnosis of disease. OBJECTIVES: To evaluate rapid, reliable, and non-destructive SERS-based diagnostic tool with multivariate data analysis including principal component analysis (PCA) and partial least square discriminant analysis (PLS-DA) for classification of different stages of typhoid on the basis of characteristic SERS spectral features. METHODS: SERS has been used for analysis of serum samples of different stages of typhoid including early acute stage and late acute stage in comparison with healthy samples, in order to investigate capability of this technique for diagnosis of typhoid. SERS spectral features associated with the biochemical changes taking place during the development of the typhoid fever were analyzed and identified. RESULTS: The value of area under the receiver operating characteristics (AUROC) for early acute stage versus healthy is 0.87 and that for healthy versus late acute stage is 0.52. PLS-DA classifier model gives values of 100 % for accuracy, sensitivity and specificity, respectively for the SERS spectral data sets of healthy versus early acute stage. Moreover, this classifier model gives values of 91 %, 89 % and 97 % for accuracy, sensitivity and specificity, respectively for the SERS spectral data sets of healthy versus late acute stage. CONCLUSIONS: Based on preliminary work it is concluded that SERS has potential to diagnose various stages of typhoid fever including early acute and late acute stage in comparison with healthy samples.

Surface-enhanced Raman spectroscopy for analysis of PCR products of viral RNA of hepatitis C patients.

In the current study, for a qualitative and quantitative study of Polymerase Chain Reaction (PCR) products of viral RNA of Hepatitis C virus (HCV) infection, surface-enhanced Raman spectroscopy (SERS) methodology has been developed. SERS was used to identify the spectral features associated with the PCR products of viral RNA of Hepatitis C in various samples of HCV-infected patients with predetermined viral loads. The measurements for SERS were performed on 30 samples of PCR products, which included three PCR products of RNA of healthy individuals, six negative controls, and twenty-one HCV positive samples of varying viral loads (VLs) using Silver nanoparticles (Ag NPs) as a SERS substrates. Additionally, on SERS spectral data, the multivariate data analysis methods including Principal Component Analysis (PCA) and Partial Least Squares Regression (PLSR) were also carried out which help to illustrate the diagnostic capabilities of this method. The PLSR model is designed to predict HCV viral loads based on biochemical changes observed as SERS spectral features which can be associated directly with HCV RNA. Several SERS characteristic features are observed in the RNA of HCV which are not detected in the spectra of healthy RNA/controls. PCA is found helpful to differentiate the SERS spectral data sets of HCV RNA samples from healthy and negative controls. The PLSR model is found to be 99% accurate in predicting VLs of HCV RNA samples of unknown samples based on SERS spectral changes associated with the Hepatitis C development.

Rapid and sensitive discrimination among carbapenem resistant and susceptible E. coli strains using Surface Enhanced Raman Spectroscopy combined with chemometric tools.

BACKGROUND: Raman spectroscopy is a powerful technique for the robust, reliable and rapid detection and discrimination of bacteria. OBJECTIVES: To develop a rapid and sensitive technique based on surface-enhanced Raman spectroscopy (SERS) with multivariate data analysis tools for discrimination among carbapenem resistant and susceptible E. coli strains. METHODS: SERS was employed to differentiate different strains of carbapenem resistant and susceptible E. coli by using silver nanoparticles (Ag NPs) as a SERS substrate. For this purpose, four strains of carbapenem resistant and three strains of carbapenem susceptible E. coli were analyzed by comparing their SERS spectral signatures. Furthermore, multivariate data analysis techniques including Principal Component Analysis (PCA), Hierarchical Cluster Analysis (HCA) and Partial Least Squares-Discriminant Analysis (PLS-DA) were performed over the spectral range of 400-1800 cm-1 (fingerprint region) for the identification and differentiation of different E. coli strains. RESULTS: The SERS spectral features associated with resistant development against carbapenem antibiotics were separated by comparing each spectrum of susceptible strains with each resistant strain. PCA and HCA were found effective for the qualitative differentiation of all the strains analysed. PLS-DA successfully discriminated the carbapenem resistant and susceptible E. coli pellets on the strain level with 99.8 % sensitivity, 100 % specificity, 100 % accuracy and 86 % area under receiver operating characteristic (AUROC) curve. CONCLUSION: SERS can be employed for the rapid discrimination among carbapenem resistant and susceptible strains of E. coil.

SERS-based viral load quantification of hepatitis B virus from PCR products.

Hepatitis B is a contagious liver disorder caused by hepatitis B virus and if not treated at an early stage, it becomes chronic and results in liver cirrhosis and hepatocellular carcinoma which can even lead to death. In present study, surface-enhanced Raman spectroscopy (SERS) is employed for the analysis of polymerase chain reaction (PCR) products of DNA extracted from hepatitis B virus (HBV) infected patients in comparison with healthy individuals. SERS spectral features are identified which are solely present in the HBV positive samples and consistently increase in intensities with increase in viral load which can be considered as a SERS spectral marker for HBV infection. For sake of understanding, these various levels of viral loads in this study are classified as low (1-1000 IU), medium (1000-10,000 IU), high (above 10,000 IU) and negative control (>1). In order to explore the efficiency of SERS for discrimination of SERS spectral datasets of different samples of varying viral loads and healthy individuals, principal component analysis (PCA) is applied. PCA is used for comparison of these classes including low, medium and high levels of viral loads with each other and with healthy class. Moreover, partial least square discriminant analysis and partial least square regression analysis are employed for the classification of different levels of viral loads in the HBV positive samples and prediction of viral loads in the unknown samples, respectively. PLS-DA is applied for validity of classification and its sensitivity and specificity was found to be 89% and 98% respectively. PLSR model was constructed for prediction of viral loads on the bases of SERS spectral markers of HBV infection with goodness value of 0.9031 and value of root means square error (RMSE) 0.2923. PLSR model also proved to be valid for prediction of blind sample.

Surface enhanced Raman spectroscopy of RNA samples extracted from blood of hepatitis C patients for quantification of viral loads.

BACKGROUND: Raman spectroscopy is a promising technique to analyze the body fluids for the purpose of non-invasive disease diagnosis. OBJECTIVES: To develop a surface-enhanced Raman spectroscopy (SERS) based method for qualitative and quantitative analysis of HCV from blood samples. METHODS: SERS was employed to characterize the Hepatitis C viral RNA extracted from different blood samples of hepatitis C virus (HCV) infected patients with predetermined viral loads in comparison with total RNA of healthy individuals. The SERS measurements were performed on 27 extracted RNA samples including low viral loads, medium viral loads, high viral loads and healthy/negative viral load samples. For this purpose, silver nanoparticles (Ag NPs) were used as SERS substrates. Furthermore, multivariate data analysis technique, Principal Component Analysis (PCA) and Partial Least Square Regression (PLSR) were also performed on SERS spectral data. RESULTS: The SERS spectral features due to biochemical changes in the extracted RNA samples associated with the increasing viral loads were established which could be employed for HCV diagnostic purpose. PCA was found helpful for the differentiation between Raman spectral data of RNA extracted from hepatitis infected and healthy blood samples. PLSR model is established for the determination of viral loads in HCV positive RNA samples with 99 % accuracy. CONCLUSION: SERS can be employed for qualitative and quantitative analysis of HCV from blood samples.

Raman spectroscopy for the qualitative and quantitative analysis of solid dosage forms of Sitagliptin.

To demonstrate the potential of Raman spectroscopy for the qualitative and quantitative analysis of solid dosage pharmacological formulations, different concentrations of Sitagliptin, an Active Pharmaceutical Ingredient (API) currently prescribed as an anti-diabetic drug, are characterised. Increase of the API concentrations induces changes in the Raman spectral features specifically associated with the drug and excipients. Principal Component Analysis (PCA) and Partial Least Squares Regression (PLSR), were used for the qualitative and quantitative analysis of the spectral responses. A PLSR model is constructed which enables the prediction of different concentrations of drug in the complex excipient matrices. During the development of the prediction model, the Root Mean Square Error of Cross Validation (RMSECV) was found to be 0.36 mg and the variability explained by the model, according to the (R2) value, was found to be 0.99. Moreover, the concentration of the API in the unknown sample was determined. This concentration was predicted to be 64.28/180 mg (w/w), compared to the 65/180 mg (w/w). These findings demonstrate Raman spectroscopy coupled to PLSR analysis to be a reliable tool to verify Sitagliptin contents in the pharmaceutical samples based on calibration models prepared under laboratory conditions.

Quantitative analysis of solid dosage forms of cefixime using Raman spectroscopy.

Quantification of antibiotics is of significant importance because of their use in the prevention and treatment of different diseases. Cefixime (CEF) is a cephalosporin antibiotic that is used against bacterial infections. In the present study, Raman spectroscopy has been applied for the identification and quantification of Raman spectral features of cefixime with different concentrations of Active Pharmaceutical Ingredient (API) and excipients in solid dosage forms. The changes in Raman spectral features of API and excipients in the solid dosage forms of cefixime were studied and Raman peaks were assigned based on the literature. Multivariate data analysis techniques including the Principal Component Analysis (PCA) and Partial Least Squares Regression analysis (PLSR) have been performed for the qualitative and quantitative analysis of solid dosage forms of cefixime. PCA was found helpful in differentiating all the Raman spectral data associated with the different solid dosage forms of cefixime. The coefficient of determination (R2), mean absolute error (MAE), and mean relative error (MRE) for the calibration data-set were 0.99, 0.72, and 0.01 respectively and for the validation data-set were 0.99, 3.15, and 0.02 respectively, that shows the performance of the model. The root mean square error of calibration (RMSEC) and root mean square error of prediction (RMSEP) were found to be 0.56 mg and 3.13 mg respectively.