Gold Standard Culture Research Articles

Tests for Early Detection of Paediatric Urinary Tract Infection

Urinary tract infection is the most common of all pediatric bacterial infections. This study aims to determine the efficacy of rapid diagnostic tests vs gram stain as against gold standard test (urine culture) and to find out the validity of each screening test for early detection of urinary tract infection in children. A cross-sectional study was conducted on children aged between 3- 15 years of suspected urinary tract infections from August 2020 to January 2021. Data collection was facilitated by using a predesigned checklist. The specimen was collected in a sterile, leak proof container by clean catch midstream technique and was subjected to urine microscopy, urine dipstick nitrite and leukocyte esterase, urine gram stain, and the gold standard test urine culture and sensitivity. Using the gold standard test, the sensitivity, specificity, positive predictive value, and negative predictive value of the other screening tests were determined. The patient's mean age was 5.16 ± 2.7 years, and half were in the age group 3-5 years. The majority of the patients were male (54%). Fever (64%) was the major symptom, followed by pain in the abdomen (52%) and dysuria (40%). Escherichia coli was the most common organism isolated (54%), followed by Klebsiella (22%). Urine gram stain has sensitivity and specificity of 95% and 100% respectively. Urinary tract infection is a common infection in children. Urine gram stain is a very sensitive and specific test for diagnosing urinary tract infections compared to other screening tests.

Seroprevalence of brucellosis and molecular characterization of Brucella spp. from slaughtered cattle in Rwanda.

Bovine brucellosis is endemic in Rwanda, although, there is a paucity of documented evidence about the disease in slaughtered cattle. A cross-sectional study was conducted in slaughtered cattle (n = 300) to determine the seroprevalence of anti-Brucella antibodies using the Rose Bengal Test (RBT), and indirect enzyme-linked immunosorbent assay (i-ELISA). Corresponding tissues were cultured onto a modified Centro de Investigación y Tecnología Agroalimentaria (CITA) selective medium and analysed for Brucella spp. using the 16S-23S ribosomal interspacer region (ITS), AMOS, and Bruce-ladder PCR assays. The seroprevalence was 20.7% (62/300) with RBT, 2.9% (8/300) with i-ELISA, and 2.9% (8/300) using both tests in series. Brucella-specific 16S-23S ribosomal DNA interspace region (ITS) PCR detected Brucella DNA in 5.6% (17/300; Brucella culture prevalence). AMOS-PCR assay identified mixed B. abortus and B. melitensis (n = 3), B. abortus (n = 3) and B. melitensis (n = 5) while Bruce-ladder PCR also identified B. abortus (n = 5) and B. melitensis (n = 6). The gold standard culture method combined with PCR confirmation identified 5.6% Brucella cultures and this culture prevalence is higher than the more sensitive seroprevalence of 2.9%. This emphasizes the need to validate the serological tests in Rwanda. The mixed infection caused by B. abortus and B. melitensis in slaughtered cattle indicates cross-infection and poses a risk of exposure potential to abattoir workers. It is essential to urgently strengthen a coordinated national bovine brucellosis vaccination and initiate a test-and-slaughter program that is not presently applicable in Rwanda.

Laboratory and field evaluation of polymerase chain reaction assays for diagnosis of zoonotic tuberculosis in bovine post mortem samples

The diagnosis of zoonotic tuberculosis revolves around detection of pathogen by gold standard culture method. In the live animals, tuberculin testing and interferon gamma assays are the options with their own limitation of specificity and cost effectiveness respectively. Clinical examination along with the affordable methods can lead to a definitive diagnosis which could help in decision making for not only the individual animal but also for the whole farm. During post-mortem examination, the post-mortem lesions with visible tubercle and acid fast staining give a fair idea of tuberculosis infected carcass for tuberculosis. The molecular testing of tissue samples with proven mycobacterial genus, Mycobacterium tuberculosis complex (which includes M. bovis also) and M. bovis species-specific primers can add to the diagnostic strength for bovine tuberculosis. In this regard, we evaluated sequential PCRs for mycobacterial genus specific primer (hsp65), M. tuberculosis complex specific primer (IS6110 & IS1081) and M. bovis specific primer (RD4 and 500 bp) in spiked and field tissue samples of cattle. The research was carried out in 2019-20 in Division of Animal Health for framing out a diagnostic confirmation protocol for TB infected cattle on tissue samples. All the PCR assays were almost equally efficient in detection of M. bovis in spiked samples, if supported by an efficient DNA extraction method which is the foremost limiting factor. It was concluded that a combination of minimum of two assays can give a definitive result along with the support of the clinical history or post mortem outcome of the animals.

Combining Complete Blood Count with Differential Count; C-Reactive Protein and Band Neutrophils in Diagnosis of Early onset Neonatal Sepsis

Aim: To assess the diagnostic accuracy of complete blood count with band versus total neutrophil ratio and C-reactive proteins as an efficient analytical procedure for determining early onset sepsis in neonates. Study design: Cross sectional analytical study Place and duration of study: Department of Paediatric Medicine, Abbas Institute of Medical Sciences Muzafarabad, AJ Kashmir from 01-02-2022 to 31-07-2022. Methodology: Fifty patients were enrolled. Coulter counter was used for analyzing leucocytes of the neonates. Thin film-smears were generated by the laboratory scientist for identifying band neutrophils separately from mature neutrophils. C-reactive proteins level <5mg/dl was considered as negative. Thio-glycate-oxide in liquid form was used for 2ml neonatal blood culture analysis mixed in the 20ml medium at 36 degree celsius. Results: There were 28 (56%) males and 22 (44%) females. The mean age of the neonates was 1.6± 0.77 days. The mean weight of the neonates was 2.4±0.51kg with a range of 4-1.2kg. A variance of band versus total neutrophil ratio was significantly higher in cultures which were positive than negative such as 70% vs 25%. There was also significant percentage of C-reactive proteins positive in positive sepsis cultures in comparison to negative blood culture. Conclusion: A combination of complete blood count with differential count, white blood cell analysis with band versus total neutrophil ratio and c-reactive proteins is an efficient early analytical procedure for determining early onset sepsis in neonates as gold standard test blood culture is time taking. Key Words: Complete blood count, C-reactive protein, Total neutrophils ratio, Early onset neonatal sepsis.

Versatile nanorobot hand biosensor for specific capture and ultrasensitive quantification of viral nanoparticles.

Accurate determination of the concentration and viability of the viral vaccine vectors is urgently needed for preventing the spread of the viral infections, but also supporting the development and assessment of recombinant virus-vectored vaccines. Herein, we describe a nanoplasmonic biosensor with nanoscale robot hand structure (Nano RHB) for the rapid, direct, and specific capture and quantification of adenovirus particles. The nanorobot allows simple operation in practical applications, such as real-time monitoring of vaccine quantity and quality, and evaluation of vaccine viability. Modification of the Nano RHB with branched gold nanostructures allow rapid and efficient assessment of human adenovirus viability, with ultrahigh detection sensitivity of only 100 copies/mL through one-step sandwich method. Nano RHB detection results were consistent with those from the gold standard median tissue culture infectious dose and real-time polymerase chain reaction assays. Additionally, the Nano RHB platform showed high detection specificity for different types of viral vectors and pseudoviruses. Altogether, these results demonstrate that the Nano RHB platform is a promising tool for efficient and ultrasensitive assessment of vaccines and gene delivery vectors.

Diagnostic Accuracy of Centor Score for Diagnosis of Group A Streptococcal Pharyngitis among Adults in Primary Care Clinics in Malaysia.

BackgroundPharyngitis is a common presentation seen in general practice, but it is difficult to differentiate whether its etiology is viral or bacterial. The Centor score gives an approximation of the etiology of the pharyngitis, which informs physicians of the need to prescribe antibiotics. This study aimed to assess the validity of the Centor score in diagnosing Group A streptococcal (GAS) pharyngitis amongst adults in Malaysia.MethodsThis cross-sectional study was conducted to compare the clinical criteria of the Centor score to the gold standard throat swab culture results amongst 215 adults presenting with sore throat in primary care clinics. The participants were adult patients who complained of sore throat and visited the three public primary care clinics in Sepang, Malaysia. The convenience sampling method was used. The throat swabs were analysed for β-haemolytic streptococci. Demographic and clinical data, including the Centor score, were analysed in relation to the pathogen.ResultsPharyngitis was diagnosed in 130 (60.5%) of the participants. Six isolates (2.4%) were identified as GAS pharyngitis. Both Centor scores 3 and 4 had a sensitivity of 50%, and specificities of 97.6% and 100%, respectively.ConclusionA Centor score < 3 is favourable for excluding a diagnosis of GAS pharyngitis. Centor scores 3 and 4 require further examination to confirm a diagnosis of GAS pharyngitis.

Rapid Detection of Bacterial Pathogens and Antimicrobial Resistance Genes in Clinical Urine Samples With Urinary Tract Infection by Metagenomic Nanopore Sequencing.

Urinary tract infections (UTIs) are among the most common acquired bacterial infections in humans. The current gold standard method for identification of uropathogens in clinical laboratories is cultivation. However, culture-based assays have substantial drawbacks, including long turnaround time and limited culturability of many potential pathogens. Nanopore sequencing technology can overcome these limitations and detect pathogens while also providing reliable predictions of drug susceptibility in clinical samples. Here, we optimized a metagenomic nanopore sequencing (mNPS) test for pathogen detection and identification in urine samples of 76 patients with acute uncomplicated UTIs. We first used twenty of these samples to show that library preparation by the PCR Barcoding Kit (PBK) led to the highest agreement of positive results with gold standard clinical culture tests, and enabled antibiotic resistance detection in downstream analyses. We then compared the detection results of mNPS with those of culture-based diagnostics and found that mNPS sensitivity and specificity of detection were 86.7% [95% confidence interval (CI), 73.5–94.1%] and 96.8% (95% CI, 82.4–99.9%), respectively, indicating that the mNPS method is a valid approach for rapid and specific detection of UTI pathogens. The mNPS results also performed well at predicting antibiotic susceptibility phenotypes. These results demonstrate that our workflow can accurately diagnose UTI-causative pathogens and enable successful prediction of drug-resistant phenotypes within 6 h of sample receipt. Rapid mNPS testing is thus a promising clinical diagnostic tool for infectious diseases, based on clinical urine samples from UTI patients, and shows considerable potential for application in other clinical infections.

Diagnosis of Bloodstream Infections: An Evolution of Technologies towards Accurate and Rapid Identification and Antibiotic Susceptibility Testing.

Bloodstream infections (BSI) are a leading cause of death worldwide. The lack of timely and reliable diagnostic practices is an ongoing issue for managing BSI. The current gold standard blood culture practice for pathogen identification and antibiotic susceptibility testing is time-consuming. Delayed diagnosis warrants the use of empirical antibiotics, which could lead to poor patient outcomes, and risks the development of antibiotic resistance. Hence, novel techniques that could offer accurate and timely diagnosis and susceptibility testing are urgently needed. This review focuses on BSI and highlights both the progress and shortcomings of its current diagnosis. We surveyed clinical workflows that employ recently approved technologies and showed that, while offering improved sensitivity and selectivity, these techniques are still unable to deliver a timely result. We then discuss a number of emerging technologies that have the potential to shorten the overall turnaround time of BSI diagnosis through direct testing from whole blood—while maintaining, if not improving—the current assay’s sensitivity and pathogen coverage. We concluded by providing our assessment of potential future directions for accelerating BSI pathogen identification and the antibiotic susceptibility test. While engineering solutions have enabled faster assay turnaround, further progress is still needed to supplant blood culture practice and guide appropriate antibiotic administration for BSI patients.

Diagnostic performance of an in-house multiplex PCR assay and the retrospective surveillance of bacterial respiratory pathogens at a teaching hospital, Kelantan, Malaysia

ABSTRACT Respiratory tract infections (RTIs), including pneumonia and pulmonary tuberculosis, are among the leading causes of death worldwide. The use of accurate diagnostic tests is crucial to initiate proper treatment and therapy to reduce the mortality rates for RTIs. A PCR assay for simultaneous detection of six respiratory bacteria: Haemophilus influenzae, Klebsiella pneumoniae, Mycobacterium tuberculosis, Pseudomonas aeruginosa, Staphylococcus aureus and Streptococcus pneumoniae, was developed in our lab. The current study aimed to evaluate the performance of this assay along with the retrospective surveillance of respiratory pathogens at a teaching hospital in Kelantan, Malaysia. Leftover sputa (n = 200) from clinical laboratories were collected and undergone DNA template preparation for PCR analysis. Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of the PCR assay were determined in comparison with the gold standard sputum culture. Overall, the accuracy performance of this assay was 94.67% (95% CI: 90.87% to 97.21%) with sensitivity, specificity, PPV and NPV of 100%, 91.67%, 87.1% and 100%, respectively. Based on the organisms detected from sputa, K. pneumoniae ranked as the top isolate (n = 48), followed by P. aeruginosa (n = 13) and H. influenzae (n = 10). Surveillance among the patients showed that the associations of bacterial positive with gender and means of acquisition were found significant (p values = 0.049 and 0.001, respectively). Besides the promising performance of this ready-to-use molecular-based assay for the rapid detection of selected bacteria pathogens, this study also highlighted significant spread of K. pneumoniae RTIs in the community.

A retrospective study of predictive value of Gram staining in the diagnosis of urinary tract infection

One of the most common infection among humans is Urinary Tract Infection that almost every person will get atleast once in their lifetime. Timely diagnosis and treatment are necessary to reduce the complications from UTI particularly among patients with comorbidities. Urine culture is considered as the gold standard diagnostic test for UTI, however it is costly and time consuming. In this study we evaluated Urine Gram stain that can be used as screening test that is also cost effective with less turn around time. The Sensitivity, Specificity, Positive predictive value (PPV) and Negative predictive value (NPV) of Urinary Gram stain were assessed with gold standard culture and sensitivity that showed the point-of-care Gram stain and pyuria can be used as rapid diagnostic test for UTI, which can be carried out quickly at low cost.

Platelet count and its indices-effectiveness in early diagnosis of neonatal sepsis

Context: Neonatal sepsis is the major cause of morbidity and mortality in developing countries requiring early diagnosis and treatment. The Gold standard Blood culture results are often delayed for 48 h with high false-negative values. Emerging evidence suggests that platelet indices such as plateletcrit, mean platelet volume (MPV), and platelet distribution width (PDW) are reliable biomarkers that are readily available while obtaining routine complete blood counts. Aim: The aim of the study was to evaluate the efficacy of platelet count (Platelets) and its indices in the early diagnosis of sepsis. Settings and Design: Blood samples were collected from all neonates admitted to the hospital with features of suspected sepsis. Materials and Methods: One hundred patients were recruited for this cross-sectional analytical prospective study. All neonates delivered in Himalayan Institute of Medical Sciences and those referred from outside with features of suspected septicemia meeting the inclusion criteria were taken for study purposes. Blood samples were collected at the time of admission. Patients were divided into three groups clinically suspected sepsis (probable sepsis), culture-positive sepsis, and culture-negative sepsis. Non-parametric tests like the Chi-square test were applied to see the association between the variables. The three groups were compared for Platelets and its indices with the Analysis of variance test. Results: MPV was 219.2, 174.8, and 205.7 (p&lt;0.031), PDW 8.3, 12.5, and 11.5 (p&lt;0.174) and MPV as 10.9, 9.94 and 11.9 (p&lt;0.556) in probable sepsis, culture-positive and culture-negative sepsis, respectively. Conclusions: Platelets and its indices can be considered as a diagnostic tool for neonatal sepsis as it is cheap, rapid, and easily available and does not require additional equipment.

Well-orchestrated physico-chemical and biological factors for enhanced secretion of osteogenic and angiogenic extracellular vesicles by mesenchymal stem cells in a 3D culture format.

The secretome of mesenchymal stem cells (MSCs) is being studied for its regenerative potential for the treatment of various disorders, including bone diseases. However, mimicking the physiological parameters of native bone could further improve MSCs' secretory profile. The proteomic analysis revealed that MSCs have a diverse secretory profile depending on the cell formats used to grow them, such as two-dimensional (2D) or three-dimensional (3D) microenvironments. Stem cells are given biochemical and biophysical stimuli in a 3D milieu that mimics in vivo situations. Compared to the gold standard monolayer culture, extracellular vesicles (EVs) released under 3D conditions improved the EV cargo numerically and qualitatively. The higher requirements of EVs in clinical trials with consistent therapeutic potential are challenging. This review discusses the impact of cell culture formats on the regenerative potential of MSCs, specifically in bone regeneration. The poor yield and heterogeneity issues have hampered the therapeutic usage of EVs. Therefore, this review further explores various engineering approaches that could enhance EVs' scalability from MSCs and their therapeutic effectiveness beyond their native utility in bone tissue regeneration. This review also highlights some of the upcoming 3D approaches/models that might be useful for the enhanced secretion of therapeutic EVs from stem cells. Finally, we discuss possible future directions and conclusions in this domain.

Molecular detection of Salmonella isolated from commercial chicken.

Salmonella in chicken, specially, the motile salmonellae, causes the food chain unsafe from farm to table and is considered a significant global threat to public health. The present study was carried out for molecular detection of Salmonellae in commercial poultry using PCR. The study was conducted for eight months, from July 2019 to February 2020, and a total of 26 poultry farms, including 15 broiler and 11-layer farms, were visited individually. Pooled faecal samples were obtained from the sheds. A total of 189 necropsy cases were examined for gastrointestinal lesions. Isolation and identification of the organism were done using microbe culture method, and the molecular characterization was performed via PCR targeting invA and ent genes. The prevalence of salmonellosis in the broiler and layer farms was recorded at 20.0% and 45.4%, respectively, through the traditional gold standard culture method. From 189 necropsy birds, salmonellosis was recorded at 1.58% dead cases. Molecular detection of Salmonella isolates by PCR targeting invA gene was confirmed in 13.33% of the broiler farms and 36.3% of the layer farms. Further detection of Salmonella enteritidis was performed by PCR targeting ent gene by which 11.11% positivity was determined. This study, focused on the Salmonella prevalence, highlighted the zoonotic importance of the bacterium in the commercial poultry farms, which can subsequently be dispersed into the human food chain causing harmful health effects.

A 9-gene biomarker panel identifies bacterial coinfections in culture-negative COVID-19 cases.

Confirmatory diagnosis of bacterial coinfections with COVID-19 is challenging due to the limited specificity of the widely used gold-standard culture sensitivity test despite clinical presentations. A misdiagnosis can either lead to increased health complications or overuse of antibiotics in COVID-19 patients. With a multi-step systems biology pipeline, we have identified a 9-gene biomarker panel from host blood that can identify bacterial coinfection in COVID-19 patients, even in culture-negative cases. We have also formulated a qPCR-based score that diagnoses bacterial coinfection with COVID-19 with the accuracy, specificity, and sensitivity of 0.93, 0.96, and 0.89, respectively. This gene signature and score can assist in the clinical decision-making process of necessary and timely prescription of antibiotics in suspected bacterial coinfection cases with COVID-19 and thereby help to reduce the associated morbidity and mortality.

Target-enriched sequencing enables accurate identification of bloodstream infections in whole blood

Bloodstream infections are within the top ten causes of death globally, with a mortality rate of up to 70%. Gold standard blood culture testing is time-consuming, resulting in delayed, but accurate, treatment. Molecular methods, such as RT-qPCR, have limited targets in one run. We present a new Ampliseq detection system (ADS) combining target amplification and next-generation sequencing for accurate identification of bacteria, fungi, and antimicrobial resistance determinants directly from blood samples. In this study, we included removal of human genomic DNA during nucleic acid extraction, optimized the target sequence set and drug resistance genes, performed antimicrobial resistance profiling of clinical isolates, and evaluated mock specimens and clinical samples by ADS. ADS successfully identified pathogens at the species-level in 36 h, from nucleic acid extraction to results. Besides pathogen identification, ADS can also present drug resistance profiles. ADS enabled detection of all bacteria and accurate identification of 47 pathogens. In 20 spiked samples and 8 clinical specimens, ADS detected at least 92.81% of reads mapped to pathogens. ADS also showed consistency with the three culture-negative samples, and correctly identified pathogens in four of five culture-positive clinical blood specimens. This Ampliseq-based technology promises broad coverage and accurate pathogen identification, helping clinicians to accurately diagnose and treat bloodstream infections.

Comparative performance of different antigens on the lateral flow assay (LFA) platform for the rapid serodiagnosis of paratuberculosis

Paratuberculosis is a globally prevalent disease, that adversely affects the economy of livestock farming. Control is largely based on early detection followed by ‘Test and Cull’ or ‘Test and Segregate’ Policy. Implementation of paratuberculosis control is a special challenge due to the non-availability of point of care diagnostics (PoCD). Therefore, the present study aimed to optimize and evaluate a lateral flow assay (LFA) for the rapid serodiagnosis of paratuberculosis in ruminant species, especially in the view of the resource-limited areas. Performance of three different antigenic preparations including native purified protoplasmic antigen (nPPA-LFA), commercial purified protoplasmic antigen (cPPA-LFA), and a cocktail of recombinant secretory proteins (RP-LFA) was evaluated as detection reagents for coating LFA strips. Comparative performance of the optimized LFA was also evaluated with gold standard tissue culture, fecal PCR (polymerase chain reaction), and plate ELISA. In addition, the onsite testing of animals belonging to different farms (endemic), species, and regions using optimized LFA was also done to highlight the on-farm testing approach. Findings revealed recombinant secretory proteins based LFA (RP-LFA) had a higher sensitivity of detection compared to other antigens. RP-LFA had a sensitivity of 77.7%, 75.44%, and 75.16% in comparison to gold standard tissue culture, fecal PCR, and plate ELISA, respectively. The specificity of RP-LFA was 100% with all reference tests. In comparison to plate ELISA, RP-LFA had a detection limit of 100% when the S/P ratio of the serum sample is ≥1.0 and 80% when the S/P ratio range of 0.8–1.0. Using RP-LFA, on-farm testing of 608 animals was done and 283 (46.5%) were found positive. Kappa analysis of present RP-LFA revealed ‘good strength of agreement’ with gold standard tissue culture, fecal PCR, and plate ELISA. Optimized RP-LFA had no cross-reactivity with bovine tuberculosis (bovine TB). The RP-LFA was found reproducible, user-friendly and test results can be interpreted within five minutes. In conclusion, the findings of the present study advocate the huge potential of LFA-based PoCD in the rapid diagnosis and control of paratuberculosis.

Clinical Trial: Magnetoplasmonic ELISA for Urine-based Active Tuberculosis Detection and Anti-Tuberculosis Therapy Monitoring.

The coronavirus disease 2019 (COVID-19) pandemic has proved the importance of fast and widespread diagnostic testing to prevent serious epidemics timely. The first-line weapon against rapidly transmitted disease is a quick and massive screening test to isolate patients immediately, preventing dissemination. Here, we described magnetoplasmonic nanozymes (MagPlas NZs), i.e., hierarchically coassembled Fe3O4–Au superparticles, that are capable of integrating magnetic enrichment and catalytic amplification, thereby the assay can be streamlined amenable to high-throughput operation and achieve ultrahigh sensitivity. Combining this advantage with conventional enzyme-linked immunosorbent assay (ELISA), we propose a MagPlas ELISA for urine-based tuberculosis (TB) diagnosis and anti-TB therapy monitoring, which enables fast (<3 h), and highly sensitive (up to pM with naked-eyes, < 10 fM with plate reader) urinary TB antigen detection. A clinical study with a total of 297 urine samples showed robust sensitivity for pulmonary tuberculosis (85.0%) and extra-pulmonary tuberculosis (52.8%) patients with high specificity (96.7% and 96.9%). Furthermore, this methodology offers a great promise of noninvasive therapeutic response monitoring, which is impracticable in the gold-standard culture method. The MagPlas ELISA showed high sensitivity comparable to the PCR assay while retaining a simple and cheap ELISA concept, thus it could be a promising point-of-care test for TB epidemic control and possibly applied to other acute infections.

Efficacy of Urine Dipstick Test in Diagnosing Urinary Tract Infection and Detection of the blaCTX-M Gene among ESBL-Producing Escherichia coli.

A urine dipstick test used for prompt diagnosis of urinary tract infection (UTI) is a rapid and cost-effective method. The main objective of this study was to compare the efficacy of the urine dipstick test with culture methods in screening for UTIs along with the detection of the blaCTX-M gene in extended spectrum β-lactamase (ESBL)-producing Escherichia coli. A total of 217 mid-stream urine samples were collected from UTI-suspected patients attending Bharatpur Hospital, Chitwan, and tested by dipstick test strip (COMBI-10SL, Germany) prior to the culture. E. coli isolates were identified by standard microbiological procedures and subjected to antimicrobial susceptibility testing by Kirby Bauer disc diffusion method following CLSI guideline. Primary screening of ESBL-producing E. coli isolates was conducted using ceftriaxone, cefotaxime and ceftazidime discs and phenotypically confirmed by combined disk diffusion test. Plasmid DNA of ESBL-producing strains was extracted by phenol-chloroform method and subjected to PCR for detection of the blaCTX-M gene. Out of 217 urine samples, 48 (22.12%) showed significant bacteriuria. Among 46 (21.20%) Gram negative bacteria recovered, the predominant one was E. coli 37 (77.08%) of which 33 (89.19%) were multidrug resistant (MDR). E. coli isolates showed a higher degree of resistance towards cefazolin (62.16%) while 81.08% of the isolates were sensitive towards amikacin followed by nitrofurantoin (70.27%). Among 14 (37.84%) phenotypically confirmed ESBL isolates, only eight (21.62%) isolates carried the blaCTX-M gene. Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of urine dipstick test were 43.75%, 77.51%, 35.59% and 82.91%, respectively. Besides, the use of dipstick test strip for screening UTI was associated with many false positive and negative results as compared to the gold standard culture method. Hence, dipstick nitrite test alone should not be used as sole method for screening UTIs.

Diagnostic performance of GeneXpert MTB/RIF assay compared to conventional Mycobacterium tuberculosis culture for diagnosis of pulmonary and extrapulmonary tuberculosis, Nepal.

Tuberculosis is an infectious disease caused by the Mycobacterium tuberculosis. It is a global health problem and major cause of death in resource-limited countries like Nepal. Timely diagnosis with sensitive testing methods could assist in early management of the disease. This study was conducted to compare the diagnostic performance of GeneXpert MTB/RIF and conventional acid-fast staining with M. tuberculosis culture. The study was carried out in the Department of Microbiology, Shree Birendra Army Hospital, Nepal. Samples (n=500) were tested with a GeneXpert MTB/RIF assay and acid-fast bacilli (AFB) smear microscopy. All samples were sent for M. tuberculosis conventional culture by the German-Nepal Tuberculosis Project, Kathmandu, Nepal (GENETUP). Out of a total 500 pulmonary and extrapulmonary samples tested, 97 samples were positive for M. tuberculosis by GeneXpert MTB/RIF assay. Out of the positive samples, only 95 samples were found positive by the culture method. The sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of AFB microscopy was 45.3%, 99.5%, 99.5% and 88.5%, respectively. The sensitivity, specificity, PPV and NPV of GeneXpert MTB/RIF was found to be 100%, 99.5%, 97.5% and 100%, respectively compared to the gold standard culture method. The GeneXpert MTB/RIF test was comparable with culture diagnosis of both pulmonary and extrapulmonary tuberculosis cases.

Bioengineered 3D electrospun nanofibrous scaffold with human liver cells to study alcoholic liver disease in vitro.

Alcohol injury induces hepatic fibrosis which gradually progresses to cirrhosis, sometimes may lead to liver cancer. Animal models are less efficient in mimicking responses of human liver cells, whereas in vitro models discussed so far are majorly based on rodent cells. In this work, a coculture of primary human hepatocytes (PHHs) with LX-2 cells was established on the unmodified (C:F_0:0), collagen-I modified (C:F_1:0), fibronectin modified (C:F_0:1) and 3:1 collagen-I to fibronectin modified (C:F_3:1) 3D electrospun fibrous scaffolds. The effect of alcohol injury was evaluated on this cell-scaffold model at 0-40μl/ml alcohol concentrations over 14days of culture period by using the gold standard sandwich culture as the control. Among all the culture groups, C:F_3:1 scaffold was able to maintain translational and transcriptional properties of human liver cells at all concentrations of alcohol treatment. The study reveals that, PHHs on C:F_3:1 were able to maintain ~4-fold and ~1.6-fold higher secretion of albumin than the gold standard sandwich culture on Day 3 and Day 7, respectively. When treated with alcohol, at concentrations of 20 and 40μl/ml, albumin secretion was also observed to be higher (~2-fold) when compared to the gold standard sandwich culture. Again as expected, in C:F_3:1 culture group on 40μl/ml alcohol treatment, albumin gene expression decreased by ~2-fold due to alcohol toxicity, whereas CYP2C9, CYP3A4, CYP2E1 and CYP1A2 gene expressions upregulated by ~3.5, ~~4, ~5 and ~15-fold, respectively in response to the alcohol injury. LX-2 cells also acquire more quiescent phenotype on C:F_3:1 scaffolds when compared to the gold standard sandwich culture upon alcohol treatment. Thus, C:F_3:1 scaffold with human liver cells was established as the potential platform to scan alcohol toxicity at varied alcohol concentrations. Thus, it can pave a promising path not only to support functional healthy human liver cells for liver tissue engineering but also to examine potential drugs to study the progression or inhibition of alcoholic liver fibrosis in vitro.