Gold Standard Culture Method Research Articles

One step for Legionella pneumophila detection in environmental samples by DNA-gold nanoparticle probe.

To develop and evaluate a DNA-gold nanoparticle (DNA-AuNP) probe assay to detect Legionella pneumophila, which causes Legionnaires' disease, compared with the gold standard culture method. Gold nanoparticles (AuNPs) were conjugated with DNA probes to detect the mip gene of L. pneumophila. The DNA-AuNP probe assay was evaluated for its specificity, sensitivity and stability. The results showed that only L. pneumophila mixed with this probe resulted in a red solution that was easily detected by the naked eye, and the colour was stable when 10mmoll-1 MgSO4 was added. The 100 Legionella isolates and 10 other bacteria led to 100% specificity. Compared with the culture method, our method showed a 100% negative predictive value, 100% sensitivity (kappa=0·87), and a detection limit of 4·5ng DNAμl-1 with a 6-min response time for the 124 colonies suspected of being Legionella. The DNA-AuNP probe reagents were stable for more than 6months. The developed DNA-AuNP probe assay has good negative predictive value, sensitivity, rapidity and ease of use, which is helpful for ruling out negative samples. The DNA-AuNP probe assay can detect the mip gene of L. pneumophila. Therefore, it may be an alternative method for screening colonies suspected of being L. pneumophila.

The Eminence of Neutrophil-lymphocyte Count Ratio in Predicting Bacteremia for Community-acquired Infections at an Emergency Medicine Department in a Tertiary Care Setting.

Introduction:The changes in the white blood cells counts and other blood parameters are well-recognized feature in sepsis. A ratio between neutrophils and lymphocytes can be used as a screening marker in sepsis. Even though new markers such as Procalcitonin and adrenomedullin have been rolled out in the field, implementation of these markers has been hindered by cost, accessibility, and proper validation. We looked for the ability of simple neutrophil-lymphocyte count ratio (NLCR) when compared to the gold standard blood culture method in predicting bacteremia, on patients presented to emergency department (ED) with features of suspected community-acquired infections.Materials and Methods:A comparative study done on 258 adult patients, admitted with suspected features of community-acquired infections. The study group included all patients who had positive blood culture results on index presentation at ED. Patients with hematological, chronic liver and retroviral diseases, patients receiving chemotherapy, and steroid medications were excluded from the study. The study group was compared with gender- and age-matched control group who were also admitted with a suspicion of the same, but in whom the blood culture results were negative.Results:There was no statistically significant difference for predicting bacteremia by NLCR (>4.63) and culture positivity methods (P = 1.00). NLCR of > 4.63 predicts bacteremia with an accuracy of 84.9%.Conclusion:In our setting, NLCR performs equally well with culture positivity, in detecting severe infection at the early phase of disease. The NLCR may, therefore, be used as a suitable screening marker at ED for suspected community-acquired infections.

PERFORMANCE OF XPERT MTB/RIF ASSAY FOR DETECTION OF M.TB IN PULMONARY AND EXTRA-PULMONARY SAMPLES IN INDIAN PATIENTS

Introduction: Conventional methods like Ziehl-Neelsen (ZN) staining and liquid culture have been the mainstay for diagnosis of Tuberculosis (TB). The gold standard Liquid Culture method has a longer turnaround time. In the wake of the TB catastrophe, newer rapid and easily accessible methods of detection are the need of the hour. A molecular method like the Xpert MTB/RIF assay has revolutionized the early and rapid diagnosis of TB. Objective of the current study was to assess the performance and utility of Xpert MTB/RIF assay for the diagnosis of M. tuberculosis in pulmonary and extra-pulmonary clinical specimens in a large Indian reference laboratory.Methodology: The reference methods used were MGIT Liquid Culture system and ZN smear microscopy. Our study was performed in Global Reference Laboratory, Metropolis Healthcare, Mumbai, India for a period of 18 months with consecutive one thousand and forty two (518 Pulmonary + 524 extra-pulmonaryspecimens) clinical specimens obtained from the patients with clinical suspicion of tuberculosis. Diagnostic performance (sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of the three methods were calculated with standard formulae.Results: In comparison to MGIT Liquid culture, sensitivity of Xpert MTB/RIF assay for pulmonary and extra pulmonary specimens were 87.18% and 68.92%, respectively while in comparison to ZN smear microscopy the sensitivity of Xpert MTB/RIF assay for pulmonary and extra pulmonary specimens were 92.67% and 83.81%, respectively.Conclusion: Our study concludes that in combination with the MGIT culture, Xpert MTB/RIF assay will significantly improve the detection rate of MTB bacteria.SAARC J TUBER LUNG DIS HIV/AIDS, 2017; XIV(1), page: 7-13

Evaluation of GeneXpert MTB/RIF for detection of Mycobacterium tuberculosis complex and rpo B gene in respiratory and non-respiratory clinical specimens at a tertiary care teaching hospital in Saudi Arabia.

ObjectivesTo assess the performance of Xpert MTB/RIF, an automated molecular test for Mycobacterium tuberculosis (MTB) and resistance to rifampin (RIF), against smear microscopy and culture method for diagnosis of MTB infection.MethodsThis is a retrospective analysis of 103 respiratory and 137 non-respiratory patient specimens suspected of tuberculosis at King Khalid University Hospital, Riyadh, Kingdom of Saudi Arabia performed between April 2014 and March 2015. Each sample underwent smear microscopy, mycobacterial culture, and GeneXpert MTB/RIF test.ResultsFifteen out of 103 respiratory samples were smear and culture positive, whereas 9 out of 137 non-respiratory samples were smear positive. Out of 9 smear positive specimens, 8 were also culture positive. All 15 culture positive respiratory samples were detected by Xpert MTB/RIF (sensitivity and positive predictive value [PPV]=100%). Similarly, all 8 culture positive non-respiratory specimens were identified by Xpert MTB/RIF (sensitivity 100%; PPV 88.8%). The Xpert MTB/RIF detected only one false positive result in 88 smear negative respiratory specimens (specificity 98.9%; negative predictive value [NPV]= 100%). All 125 smear negative non-respiratory specimens tested negative by culture and Xpert MTB/RIF (sensitivity, specificity, PPV, NPV= 100%).ConclusionThe performance of Xpert MTB/RIF was comparable to the gold standard culture method for identification of MTB in both respiratory and non-respiratory clinical specimens.

Molecular surveillance on Streptococcus pneumoniae carriage in non-elderly adults; little evidence for pneumococcal circulation independent from the reservoir in children.

Carriage of Streptococcus pneumoniae in adults is rarely detected by the gold standard culture method. With molecular tests of high sensitivity now available, we analysed upper respiratory tract samples collected during autumn/winter 2012/2013 from parents of PCV7-vaccinated infants and from childless adults, directly comparing culture and qPCR-based S. pneumoniae detection. As compared to the gold standard of testing nasopharyngeal swabs, qPCR-based analysis of oral samples significantly improved detection of pneumococcal carriage (5% versus 20%, p < 0.0001) with higher carriage rates in parents compared to childless adults (34% versus 7%; p < 0.001). Molecular methods also increased the number of serotype-carriage events detected with higher carriage frequencies of serotypes 3 and 7A/F and lower of serotypes 6C/D and 15A/B/C in parents compared to their infant children. We provide evidence that culture-based methods severely underestimate adult carriage rates and for the superiority of testing oral samples over nasopharyngeal swabs. The substantial circulation of pneumococci in parents is however, not representative for the entire adult population. While age-associated differences in serotype carriage suggests reservoirs outside infants as potential sources of vaccine-serotypes contributing to weakening of vaccine herd effects, we find no evidence for reservoirs in adults contributing to serotype replacement in carriage.

Novel potential diagnostic test for Mycobacterium tuberculosis complex using combined immunomagnetic separation (IMS) and PCR-CTPP

To exploit immunomagnetic separation combined with PCR with confronting two-pair primers (IMS-PCR-CTPP) as a rapid method for detection of Mycobacterium tuberculosis complex (MTC) and identification of Mycobacterium bovis from sputum specimens. Monoclonal antibody (mAb) against the mycobacterial antigen, 85B (Ag85B), was coupled with magnetic particles for specific immunomagnetic separation (IMS) of Mycobacterium spp. Immunofluorescence assay indicated the capability of mAb to bind to Ag85B in both the recombinant and the native form. The IMS combined with PCR-CTPP targeting the mycobacterial lep B gene was further implemented using 133 sputum samples with acid-fast bacilli grading from negative to 3+. The results showed the sensitivity and specificity of IMS-PCR-CTPP vs gold standard culture method were 89·9 and 88·6% respectively. The IMS-PCR-CTPP method shortens the time for tuberculosis (TB) diagnosis from months to a day. This method is also suitable for investigation of MTC and epidemiological study of Myco.bovis in sputum specimens. This study is the first report emphasizing the combination of IMS and PCR-CTPP for the detection of MTC and simultaneous identification of Myco.bovis from sputum. It could be used for TB diagnosis in resource-limited countries with high TB burden.

Detection of foodborne bacterial zoonoses by fluorescence in situ hybridization

The efficient and timely detection of bacterial pathogens remains a major public health concern throughout the world. Fluorescence in situ hybridization (FISH) is a promising tool to detect bacteria since it incorporates the advantages of rapid detection methods with the live/dead differentiation capacity of the gold standard culture methods. However, multiplexing pathogen detection, weak FISH signals and the establishment of a quantitative and sensitive direct enumeration approach remain troublesome obstacles for a widespread use in food microbiology. Therefore, we developed and tested a comprehensive set of highly specific multiplex-FISH tests for the simultaneous detection of various foodborne bacterial zoonoses, including important pathogens like Salmonella enterica, thermophilic Campylobacter and Listeria monocytogenes. The detection of thermophilic Campylobacter spp., the most frequent bacterial zoonosis in the EU, in artificially spiked chicken breast by FISH proved to be as sensitive as the conventional ISO standard, but results were available much earlier. Strongly enhanced FISH signals for Campylobacter spp., enabling detection in matrices with high background fluorescence, were accomplished by employing several probes for this target group. For the direct detection of bacteria, independent of cultural enrichment, filtration proved to be appropriate although this method is less sensitive and thus primarily suitable for higher bacterial loads. The type of membrane filter as well as the fluorescence channel significantly influenced the efficiency of detection. Furthermore, the implementation of GFP-expressing bacteria as a quantitative standard allowed the enumeration of target pathogens after filtration. In summary, our results demonstrate the applicability of FISH for food microbiology and offer new solutions for prevalent problems in FISH-testing.

Assessment of Rapid Dipstick Test for Diagnosis of Urinary Tract Infection in Asymptomatic Pregnant Female

&lt;strong&gt;Background:&lt;/strong&gt;Bacteriuria during pregnancy has been known to cause many complications like low birth weight and premature delivery.&lt;p&gt;&lt;strong&gt;Objective&lt;/strong&gt;: This study was done to evaluate the diagnostic accuracy of rapid dipstick test to predict urinary tract infection in pregnancy against the gold standard urine culture.&lt;/p&gt;&lt;p&gt;&lt;strong&gt;Material&amp;amp;Methods:&lt;/strong&gt; A total of 200 mid stream urine samples were collected from asymptomatic pregnant females. These specimens were cultured in blood agar and MacConkey's agar by using the standard loop technique and incubated aerobically at 37°C overnight. The criterion for clinically significant bacteriuria was either a pure or predominant culture of &amp;gt;10&lt;sup&gt;5&lt;/sup&gt; colony forming units (CFU)/ml. All the specimens were also examined microscopically for pyuria and bacteriuria.&lt;/p&gt;&lt;p&gt;&lt;strong&gt;Results:&lt;/strong&gt; The prevalence of asymptomatic bacteriuria in pregnancy was 15 % in our study. The sensitivity and the specificity for leucocyte esterase were 85.7% and 74.4% and for nitrites, they were 64.2% and 72%.&lt;/p&gt;&lt;p&gt;&lt;strong&gt;Conclusion:&lt;/strong&gt; The study revealed that use of either leukocyte esterase or nitrite for screening of asymptomatic bacteriuria in pregnancy was associated with many false positive and negative results when compared with the gold standard urine culture method. By using their combination maximum negative predictive value of .98 was achieved.&lt;/p&gt;

THE ANTI-TB DRUG SENSITIVITY OF Mycobacterium tuberculosis FROM CEREBROSPINAL FLUID AND BONE TISSUE BIOPSY SPECIMENS OF PATIENTS SUSPECTED TUBERCULOUS MENINGITIS AND SPINAL TB IN Dr SOETOMO HOSPITAL INDONESIA

Tuberculous meningitis (TBM) is an infection of meningens which potentially life threatening with significant morbidity and mortality. Spinal TB has the same problem with TBM, infection in bone and joint, the delayed diagnosis worsens the prognosis. The rapid and accurate diagnosis plus promt adequate treatment is essential for the good outcome. The aim of this research is to study the first line drug sensitivity of Mycobacterium tuberculosis isolated from specimens of cerebrospinal fluid from suspected tuberculous meningitis patients and bone tissue biopsy from suspected spinal TB patients. The method of this research is TB Laboratory examination in Department of Clinical Microbiology – Dr. Soetomo General Hospital, Indonesia, using the gold standard liquid culture method MGIT 960 System (Becton Dickinson) and solid culture method with Lowenstein-Jensen medium. The specimens CSF from 50 TBM patients at January 2013 until May 2014. Positive isolate detection of Mycobacterium tuberculosis complex were 11 isolates (22%), which sensitivity 100% (11/11 isolates) to Rifampin (R), Pyrazinamide (Z), Ethambutol (E), and Streptomycin (S); one isolate resistant to Isoniazid, sensitivity to Isoniazid 90,90% (10/11); and received 21 specimens of bone tissue biopsy which positive 5 isolates (23%), all isolates sensitive 100% (5/5 isolates) to Rifampin and Pyrazinamide, and 1 isolates resistant to Isoniazid, Ethambutol, and Streptomycin, in which sensitivity 80% (4/5 isolates) to Isoniazid, Ethambutol, and Streptomycin. The conclusion of this research is positivity detection 22% of CSF specimens, and 23% of bone tissue biopsy were low. All isolates sensitive 100% to Rifampin and Pyrazinamide, and 80-90% sensitive to Isoniazid.

Evaluating the Auto-MODS Assay, a Novel Tool for Tuberculosis Diagnosis for Use in Resource-Limited Settings

There is an urgent need for simple, rapid, and affordable diagnostic tests for tuberculosis (TB) to combat the great burden of the disease in developing countries. The microscopic observation drug susceptibility assay (MODS) is a promising tool to fill this need, but it is not widely used due to concerns regarding its biosafety and efficiency. This study evaluated the automated MODS (Auto-MODS), which operates on principles similar to those of MODS but with several key modifications, making it an appealing alternative to MODS in resource-limited settings. In the operational setting of Chiang Rai, Thailand, we compared the performance of Auto-MODS with the gold standard liquid culture method in Thailand, mycobacterial growth indicator tube (MGIT) 960 plus the SD Bioline TB Ag MPT64 test, in terms of accuracy and efficiency in differentiating TB and non-TB samples as well as distinguishing TB and multidrug-resistant (MDR) TB samples. Sputum samples from clinically diagnosed TB and non-TB subjects across 17 hospitals in Chiang Rai were consecutively collected from May 2011 to September 2012. A total of 360 samples were available for evaluation, of which 221 (61.4%) were positive and 139 (38.6%) were negative for mycobacterial cultures according to MGIT 960. Of the 221 true-positive samples, Auto-MODS identified 212 as positive and 9 as negative (sensitivity, 95.9%; 95% confidence interval [CI], 92.4% to 98.1%). Of the 139 true-negative samples, Auto-MODS identified 135 as negative and 4 as positive (specificity, 97.1%; 95% CI, 92.8% to 99.2%). The median time to culture positivity was 10 days, with an interquartile range of 8 to 13 days for Auto-MODS. Auto-MODS is an effective and cost-sensitive alternative diagnostic tool for TB diagnosis in resource-limited settings.

Diagnostic accuracy of rapid urine dipstick test to predict urinary tract infection among pregnant women in Felege Hiwot Referral Hospital, Bahir Dar, North West Ethiopia.

BackgroundUntreated bacteriuria during pregnancy has been shown to be associated with low birth-weight and premature delivery. Therefore, routine screening for bacteriuria is advocated. The decision about how to screen pregnant women for bacteriuria has always been a balance between the cost of screening versus the sensitivity and specificity. This study was designed to evaluate the diagnostic accuracy of the rapid dipstick test to predict urinary tract infection in pregnancy against the gold standard urine culture.MethodA total of 367 mid stream urine samples were collected, inoculated on MacConkey, Manitol salt agar (MSA) and blood agar and incubated aerobically at 37°C for overnight. Specimens were classified as “positive” for urinary tract infection (UTI) if the growth of the pathogen(s) was at a count ≥ 105 colony-forming units per milliliter (cfu/mL) of urine and classified as “negative” with growth of <105 cfu/mL. Urine samples were tested for the presence of nitrite and leukocyte esterase using dipstick rapid test in accordance to the manufacturer’s instructions.ResultsFrom the total study participants, 37 pregnant women were symptomatic and the remaining 330 pregnant women were asymptomatic. The sensitivity and specificity of dipstick tests of leukocyte esterase was 50% and 89.1% for pregnant women with asymptomatic UTI(ABU) and 71.4% and 86.7% for symptomatic UTI respectively and for nitrite 35.7% and 98.0% for ABU and 57.1% and 96.7% symptomatic UTI.ConclusionThis study revealed that the use of dipstick leukocyte esterase and nitrite for screening UTI particularly asymptomatic bacteriuria was associated with many false positive and negative results when it was compared against the gold standard culture method. The low sensitivity and positive predictive value of urine dipstick test proved that culture should be used for the diagnosis of UTI.

Establishment of an in-House Loop-Mediated Isothermal Amplification (LAMP) for a Rapid Detection of S almonella Typhi and S almonella Paratyphi A at Low-Resource Settings

An in-house loop-mediated isothermal amplification (LAMP) method was established for the detection of Salmonella Typhi and Salmonella Paratyphi A using primers that were designed based on gene coding for putative regulatory protein in S. Typhi (STY4220) and S. Paratyphi A (SSPA3213). This established in-house LAMP assay gave positive results to all the 14 S. Typhi and 20 S. Paratyphi A isolates used in this study and negative to 20 other Salmonella and 19 non-Salmonella bacteria. When tested on 60 clinical samples, it gave positive results to 10 clinical samples containing either S. Typhi or S. Paratyphi A and negative to the other 50 samples, which were similar to those results obtained by gold standard culture method. Because of its rapidity and simplicity, this LAMP offers a promising method for the detection of both S. Typhi and S. Paratyphi A for screening purposes at resource-limited settings. Practical Applications Current detection methods for S. Typhi and S. Paratyphi A have several limitations; culture method is time-consuming and laborious, while polymerase chain reaction-based methods require expensive equipments and trained personnel which are usually not available in the low-resource setting. Therefore, LAMP established in this study offers a simple, rapid, sensitive and specific method for the screening of S. Typhi and S. Paratyphi A especially at resource-limited settings.

Detonation Nanodiamonds for Rapid Detection of Clinical Isolates of Mycobacterium tuberculosis Complex in Broth Culture Media

Routinely used molecular diagnostic methods for mycobacterium identification are expensive and time-consuming. To tackle this problem, we develop a method to streamline identification of Mycobacterium tuberculosis complex (MTBC) in broth culture media by using detonation nanodiamonds (DNDs) as a platform to effectively capture the antigen secreted by MTBC which is cultured in BACTEC MGIT 960, followed by the analysis of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF MS). The 5 nm DNDs can capture the MTBC secretory antigen without albumin interference. With on diamond digestion, we confirm the DND captured antigen is cell filtrate protein 10 (CFP-10) because its Mascot analysis shows a score of 68. The dot blotting method further verifies a positive reaction with anti-CFP-10, indicating that CFP-10 is secreted in the medium of mycobacterium growth indicator tube (MGIT) and captured by DNDs. The minimal CFP-10 protein detection limit was 0.09 μg/mL. Furthermore, our approach can avoid the false-positive identification of MTBC by immunological methods due to cross-reactivity. Five hundred consecutive clinical specimens subjected to routine mycobacteria identification in hospital were used in this study, and the sensitivity of our method is 100% and the specificity is 98%. The analysis of each MTBC sample from culture solution can be finished within 1 h and thus shortens the turnaround time of MTBC identification of gold standard culture methods. In sum, DND MALDI-TOF MS for the detection of MTBC is rapid, specific, safe, reliable, and inexpensive.

A new 24 h ELISA culture based method for Helicobacter pylori chemosusceptibility

BackgroundClarithromycin (CH) and metronidazole (MZ) are routinely used in Helicobacter pylori treatment regimes. Recently, treatment with these antibiotics has been reported to fail in >30% of patients due to increasing...