Gold Standard Culture Method Research Articles

Use of in vivo confocal microscopy in suspected Acanthamoeba keratitis: a 12-year real-world data study at a Swedish regional referral center

PurposeTo report real-world data (RWD) on the use of in vivo confocal microscopy (IVCM) in handling cases of suspected Acanthamoeba keratitis (AK) cases at a regional referral center during a 12-year period.MethodsRetrospective study of patients with suspected AK presenting at a regional referral center for IVCM in Sweden from 2010 to 2022. Demographics, symptoms, outcomes, and clinical management were analyzed, and IVCM images were interpreted.ResultsOf 74 included patients with suspected AK, 18 (24%) were IVCM-positive, 33 (44%) were IVCM-negative, 15 had inconclusive IVCM results (20.2%), and 8 (11%) were referred for a second opinion based on IVCM, 4 of which were IVCM-positive (5.5%), yielding an overall IVCM-positive rate of 29.5%. Cultures were taken in 38 cases (51%) with only 2 cases (2.7%) culture-positive for AK. Of IVCM-negative cases, cultures were taken in 22 (67%) of cases and 100% of these were AK-negative. IVCM-positive cases had more clinic visits (median 30, P = 0.018) and longer follow-up time (median 890 days, P = 0.009) than IVCM-negative patients, while visual acuity improvement did not differ (P > 0.05). Of IVCM-positive cases, 10 (56%) underwent surgery despite prior anti-amoebic treatment, and 14 (78%) had 3 or more IVCM examinations during follow-up, with cysts (100%), dendritic cells (89%) and inflammatory infiltrate (67%) as the most prevalent features. Longitudinal IVCM indicated improvement in cysts, dendritic cells and subbasal nerves with treatment, while clinical resolution was not always consistent with complete absence of cysts.ConclusionsIn a real-world setting, IVCM has a high reliability in classifying AK-negative cases, while IVCM detects AK-positive cases more frequently than the gold-standard culture method, leading to its preferential use over the culture method where time or resources are limited. Despite this, a subset of cases are IVCM-inconclusive, the clinical course of referred patients is long requiring many hospital visits, and visual acuity in most cases does not improve with medical treatment alone. Information sharing across centers and standardization of referral and diagnostic routines is needed to exploit the full potential of IVCM in AK patient management.

Rapid Minimum Inhibitory Concentration (MIC) Analysis Using Lyophilized Reagent Beads in a Novel Multiphase, Single-Vessel Assay.

Antimicrobial resistance (AMR) is a global threat fueled by incorrect (and overuse) of antibiotic drugs, giving rise to the evolution of multi- and extreme drug-resistant bacterial strains. The longer time to antibiotic administration (TTA) associated with the gold standard bacterial culture method has been responsible for the empirical usage of antibiotics and is a key factor in the rise of AMR. While polymerase chain reaction (PCR) and other nucleic acid amplification methods are rapidly replacing traditional culture methods, their scope has been restricted mainly to detect genotypic determinants of resistance and provide little to no information on phenotypic susceptibility to antibiotics. The work presented here aims to provide phenotypic antimicrobial susceptibility testing (AST) information by pairing short growth periods (~3-4 h) with downstream PCR assays to ultimately predict minimum inhibitory concentration (MIC) values of antibiotic treatment. To further simplify the dual workflows of the AST and PCR assays, these reactions are carried out in a single-vessel format (PCR tube) using novel lyophilized reagent beads (LRBs), which store dried PCR reagents along with primers and enzymes, and antibiotic drugs separately. The two reactions are separated in space and time using a melting paraffin wax seal, thus eliminating the need to transfer reagents across different consumables and minimizing user interactions. Finally, these two-step single-vessel reactions are multiplexed by using a microfluidic manifold that allows simultaneous testing of an unknown bacterial sample against different antibiotics at varying concentrations. The LRBs used in the microfluidic system showed no interference with the bacterial growth and PCR assays and provided an innovative platform for rapid point-of-care diagnostics (POC-Dx).

Evaluation of an automated rapid urine culture method for urinary tract infection: Comparison with gold standard conventional culture method.

Urinary tract infection is one of the most prevalent disease affecting people from all age groups. For its diagnosis, conventional culture and antibiotic susceptibility is the gold standard. However, its major limitation is that the results take minimum of 24​h and antibiotic susceptibility is available after 48​h. Automated culture methods having comparable sensitivity and specificity as compared to conventional culture should be evaluated for routine diagnostics. With this aim we evaluated the diagnostic accuracy of automated urine culture method HB&L uroquattro by comparing with the gold standard conventional culture method. A total of 1220 urine samples were included in the study. Semi-quantitative urine culture was performed using standard methods on cysteine lactose electrolyte deficient medium. For the automated culture, HB&L Uroquattro (Alifax, Polverara, PD, Italy), standard guidelines given in the manual of the instrument were followed. Diagnostic performance in terms of sensitivity, specificity, positive and negative likelihood ratios, positive and negative predictive values were calculated. Based on the final interpretation of conventional culture for the total 1220 samples, 26 samples (2.1%) showed major non-concordance as they were identified as sterile by HB&L but had significant growth by conventional culture and 19.9% showed minor non-concordance. At 100-999 colony forming unit/ml, HB&L has high negative predictive value i.e. 96.6% with 95% CI (95.2%-97.6%) and sensitivity i.e. 92.66% with 95% CI of (89.42%-95.15%). The HB&L Uroquattro seems to be a reliable instrument to obtain urine microbiological results in a timely manner. This technique can give presumptive report to the clinician within 5​h only for initiation of empirical antibiotics in cases of positive results.

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.

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.

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.

Rapid macrolide and amikacin resistance testing for Mycobacterium abscessus in people with cystic fibrosis.

Introduction. Mycobacterium abscessus complex (MABSC) is an environmental organism and opportunistic pathogen. MABSC pulmonary infections in people with cystic fibrosis are of growing clinical concern. Resistance data guide the use of macrolides and amikacin in MABSC pulmonary disease treatment. MABSC can acquire resistance against macrolides or amikacin via 23S or 16S rRNA gene mutations, respectively.Gap Statement. Current culture-based methods for MABSC detection and antibiotic resistance characterization are typically prolonged, limiting their utility to directly inform treatment or clinical trials. Culture-independent molecular methods may help address this limitation.Aim. To develop real-time PCR assays for characterization of key 23S or 16S rRNA gene mutations associated with constitutive resistance in MABSC.Methodology. We designed two real-time PCR assays to detect the key 23S and 16S rRNA gene mutations. The highly conserved nature of rRNA genes was a major design challenge. To reduce potential cross-reactivity, primers included non-template bases and targeted single-nucleotide polymorphisms unique to MABSC. We applied these assays, as well as a previously developed real-time PCR assay for MABSC detection, to 968 respiratory samples from people with cystic fibrosis. The results from the molecular methods were compared to those for gold standard culture methods and 23S and 16S rRNA gene sequencing.Results.The real-time PCR MABSC detection assay provided a sensitivity of 83.8 % and a specificity of 97.8 % compared to culture. The results from the real-time PCR resistance detection assays were mostly concordant (>77.4 %) with cultured isolate sequencing. The real-time PCR resistance detection assays identified several samples harbouring both resistant and susceptible MABSC, while culture-dependent methods only identified susceptible MABSC in these samples.Conclusion. Using the molecular methods described here, results for health care providers or researchers could be available days or weeks earlier than is currently possible via culture-based antibiotic susceptibility testing.

Evaluation of the Performance of Widal Slide Agglutination Test Compared to Blood Culture and Evaluation of Interferon Gamma Response in the Diagnosis of Typhoid Fever

Typhoid fever remains a public health challenge in developing countries including Nigeria. Widal test is widely used for the diagnosis of typhoid fever due to its simplicity and short turnaround time. However, the specificity of this test has been debated. The aim of the study was to evaluate the performance of Widal test compared to blood culture and determine interferon gamma response among the study subjects. Blood samples were collected from 90 patients who complained of fever and other symptoms suggestive of typhoid fever. Widal slide agglutination test, automated blood culture and interferon gamma concentrations were conducted using rapid antibody detection kit, BACTEC and sandwich enzyme linked immunosorbent assay (ELISA) respectively. Of the 90 samples tested, 63 (70.0%) were positive for anti-Typhi O antigen while 42 (46.7%) were positive for anti-Typhi H antigen. Similarly, 18 (20%) of the blood samples were non- S. Typhi culture positive while 72 (80%) had no bacteria isolated. None of the cases had S. Typhi positive culture. With regards to interferon gamma, subjects with lower levels of 15pg/mL had no bacteria isolated from their blood. As the interferon gamma concentration increased, more subjects had non- S. Typhi bacteria isolated from their blood which shows the relationship between interferon gamma and bacteraemia. The study demonstrated that the use of Widal serology test in the diagnosis of typhoid fever may be erroneous as all the samples were found to be negative for S. Typhi using the gold standard culture methods while Interferon gamma concentration was statistically related to the isolation of non- S. Typhi in blood culture as such, could be a good marker for the development of an alternative screening test, possibly an interferon gamma based detection system for typhoid fever. However, further research is recommended to elucidate that.

Identification of Bacterial and Fungal Pathogens by rDNA Gene Barcoding in Vitreous Fluids of Endophthalmitis Patients

ABSTRACT Purpose: To identify the bacterial and fungal pathogens in ocular samples of clinically suspected endophthalmitis patients by conventional culture methods and 16S and 28S rDNA gene sequencing respectively. Methods: A total of 88 patients with clinically suspected endophthalmitis were included in this study. Under sterile operating conditions, a vitreous fluid (0.1–0.2 ml) was obtained by pars plana vitrectomy procedure. The samples were processed for conventional microbiology methods and PCR. PCR targeting 16S rDNA gene for bacteria and 28S rDNA gene for fungus were performed individually using the MightyAmp DNA Polymerase Ver. 2 (TaKaRa China) kit. The PCR amplified samples were sequenced and aligned using CLUSTAL-W tool. The phylogenetic tree was constructed by Neighborhood joining along with the reference sequences downloaded from NCBI database using MEGA X software. Results: 67 Post-operative, 12 Endogenous and 9 traumatic endophthalmitis patients were included as study subjects. By the direct culturing bacterial growth was observed in 17 samples and fungal growth in three samples. PCR was positive for all the culture positive samples, in addition 14 were positive in culture negative samples. The predominant species identified in gram-positive bacteria were Staphylococcus spp., and Pseudomonas spp. in the gram-negative group. Both PCR and culture identified only three samples positive for fungal pathogens which were identified as Aspergillus fumigatus, Candida albicans, and Exerohilum rostratum. Conclusions: PCR based molecular diagnosis is more sensitive than the conventional gold standard culture methods in endophthalmitis. Bacterial pathogens were found to be the predominant in causing endophthalmitis than fungal pathogens.

Onion-like carbon re-inforced electrospun polyacrylonitrile fibres for ultrasensitive electrochemical immunosensing of Vibrio cholerae toxin

This work describes the use of electrospun onion-like carbon-polyacrylonitrile fibre (OLC-PAN) hybrids as an efficient electrode platform for electrochemical immunosensor for Vibrio cholerae toxin in water samples. First, the OLC-PAN nanohybrid was obtained by electrospinning technique. The immunosensor was constructed by covalent integration of the V. cholerae antibody on OLC-PAN hybrid-based glassy carbon electrode. The electrochemical detection of the antigen of the V. cholerae present in water samples was studied using the square wave voltammetry (SWV). The OLC-PAN-based electrode, unlike those of the individual components (OLC and PAN), showed excellent electrochemical sensing properties: as ultralow detection limit (LoD ≈ 2.5 × 10−17 g mL−1) and quantification limit (LoQ ≈ 3 × 10−15 g mL−1) and wide linear concentration range (10−13–10−5 g mL−1). The OLC-PAN-based electrochemical immunosensor was used to test for real water samples suspected to be infected with cholera, and successfully validated with both the gold-standard culture method and the polymerase chain reaction (PCR) technique. The PCR method showed poor sensitivity (LoD ≈ 5.0 × 10−7 g mL−1) compared with the SWV, proving the extremely high sensitivity and low-cost advantages of the electrochemical techniques over the conventional PCR. This work reveals the unique synergy between OLC and PAN that can be harnessed for potential development of high-performance electrochemical immunosensors for cholera disease in resource-limited communities.

A smart microfluidic platform for rapid multiplexed detection of foodborne pathogens

Rapid and sensitive detection of foodborne pathogens in food industry is of high importance in day-to-day practice to ensure safe food. To address this issue, multiple foodborne pathogens are targeted for rapid identification based in DNA amplification. A 3D PDMS sponge was fabricated using salt crystals as scarifying mold and functionalized with a ligand, apolipoprotein-H (ApoH), to test bacterial capturing for both Gram positive (L. monocytogenes) and negative bacteria (Salmonella spp.), in a microfluidic device. Pure culture of both pathogens in a range of ~10–105 CFU/mL were tested and the application of the developed automated pre-concentration protocol in real samples was verified using spiked surface samples after swab sampling. Bacterial DNA was extracted directly from the sponge and used for Real Time quantitative Polymerase Chain Reaction (qPCR) detection. The sponges did not show any significant resistance to sample flow and could easily be incorporated in a microfluidic device. A capture efficiency above 70% was observed for both targeted (Gram positive and Gram negative) pathogens and a Limit of Detection (LoD) in the range of 103 and 104 CFU/mL was obtained for Salmonella spp. and L. monocytogenes, respectively. Using this approached, we are able to perform multiplexed (Gram positive and Gram negative) capturing and reduce the enrichment time compared to the gold standard plate culture (over 1-day) method. The use of a 3D sponge for direct capturing of multiplexed pathogen on microfluidic device, followed by qPCR detection is an efficient and versatile method to stratify the presence of bacteria. This approach and methodology has potential to be integrated in full automatized device and used as point of need (PoN) system for foodborne pathogen stratification in food packaging/production industries.

Combining impedance biosensor with immunomagnetic separation for rapid screening of Salmonella in poultry supply chains

Salmonella screening is a key to ensure food safety in poultry supply chains. Currently available Salmonella detection methods including culture, polymerase chain reaction and enzyme-linked immuno-sorbent assay could not achieve rapid, sensitive, and in-field detection. In this study, different strategies for separation and detection of Salmonella were proposed, compared, and improved based on our previous studies on immunomagnetic separation and impedance biosensor. First, the coaxial capillary for immunomagnetic separation of target bacteria was improved with less contamination, and 3 strategies based on the improved capillary and immunomagnetic nanoparticles were compared to separate the target bacteria from sample and form the magnetic bacteria. The experimental results showed that the strategy of capture in tube and separation in capillary was the most suitable with separation efficiency of approximately 88%. Then, the immune gold nanoparticles coated with urease were used to label the magnetic bacteria, resulting in the formation of enzymatic bacteria, which were injected into the capillary. After the urea was catalyzed by the urease on the enzymatic bacteria in the capillary, different electrodes were compared to measure the impedance of the catalysate and the screen-printed electrode with higher sensitivity and better stability was the most suitable. This impedance biosensor-based bacterial detection strategy was able to detect Salmonella as low as 102 CFU/mL in 2 h without complex operations. Compared to the gold standard culture method for practical screening of Salmonella in poultry supply chains, this proposed strategy had an accuracy of approximately 90% for 75 real poultry samples.

Nanoparticle-Based Biosensing Assay for Universally Accessible Low-Cost TB Detection with Comparable Sensitivity as Culture.

Tuberculosis (TB) is the leading cause of death globally, surpassing HIV. Furthermore, multidrug-resistant and extensively drug-resistant TB have become global public health threats. Care of TB patients starts with quality, accessible, and affordable diagnosis. The study presents a novel technique called nanoparticle-based colorimetric biosensing assay (NCBA) based on the principles of magnetically activated cell enrichment. A total of 1108 sputum samples were subjected to sputum smear microscopy (SSM), NCBA, and standard culture. SSM and NCBA were completed in 20 min; culture was completed in 8 weeks. Results show that NCBA has matching sensitivity of 100.0% and specificity of 99.7% compared to the gold standard culture method at a cost of $0.50/test based on Peruvian conditions. Sputum smear microscopy has 63.87% sensitivity compared to culture. NCBA has the potential of being used in local health clinics as it only requires a microscope that is widely available in many rural areas. Because NCBA could detect low levels of bacterial load comparable to culture, it could be used for rapid and early TB-onset detection. The gain in time is critical as TB is airborne and highly infectious, minimizing contact exposure. Early detection could lead to early treatment, while the patient’s immune system is still high. The low cost makes NCBA affordable and accessible to those who need them the most.

The yield of Auramine O staining using led microscopy with bleach treated sputum samples for detection of pulmonary tuberculosis at St. Peter tuberculosis specialized hospital, Addis Ababa, Ethiopia

BackgroundSmear microscopy is the mainstay for diagnosis of Tuberculosis (TB) in Ethiopia. This technique; however, is insensitive to detect Mycobacteria from most clinical specimens. Currently, light emitting diode (LED) fluorescence microscope is advocated to be used in high Tuberculosis (TB) burden settings by World Health Organization (WHO). However, the utility of this method is not evaluated for bleach treated sputum samples in Ethiopia. ObjectiveThe objective of the study is to evaluate the diagnostic importance of Auramine O (AO) staining in direct and concentrated sputum against conventional Zehil-Neelsen (ZN) and culture from the sputum samples of suspected pulmonary tuberculosis patients. MethodsA cross-sectional study was conducted on 346 adult new pulmonary TB suspected patients at St. Peter's Specialized Hospital, Addis Ababa, Ethiopia. Three sputum samples (spot-morning-spot) were collected in sterile cups for direct Zehil-Neelsen and AO staining. Morning sputum samples were used for Mycobacterial culture on Mycobacterial Growth Indicator Tube (MGIT) 960. Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) were evaluated against the gold standard culture method. Data were analyzed using STATA version 13.0. All statistical tests were considered as statistically significant if the two sided P-value was < 0.05. ResultsBleach treated sputum samples with AO staining yielded more cases as compared to direct ZN and direct AO by 6.3% and 11.5%, respectively. The sensitivity of concentrated AO and direct AO were remarkably high as compared to conventional ZN (71.8% vs. 44.5% and 62.7% vs. 44.5%). The concentrated sputum with staining of AO had a high rate (18.6%) of detecting scanty graded smears as compared to conventional ZN method. ConclusionsOur findings indicated that the concentrated sputum with AO staining yielded high rate of sensitivity (71.8%) as compared to the conventional ZN method (44.5%). Moreover, the concentrated sputum with AO staining had superior ability in detecting scanty graded smears compared to the conventional ZN method. Therefore, it is recommended to utilize AO staining with LED microscopy for better diagnosis of Acid Fast Bacilli (AFB) from TB suspected cases and patients with pauci-bacillary TB in Ethiopia.

Mixed Multiplex Allele-Specific PCR (MiMAS-PCR) test for rapid detection of MDR and XDR-TB from the sputum of pulmonary TB patients

Until now, Multi-Drug Resistant TB (MDR-TB) and Extended Drug-Resistant TB (XDR-TB) are still challenging to detect rapidly, since the gold standard culture method takes at least eight weeks to achieve diagnostic results. In this research, we designed a rapid and accurate detection method to detect MDR and XDR-TB using a technique which identifies specific genes encoding susceptibility to first and second-line anti-TB drugs combined in a PCR technique called Mixed Multiplex Allele-Specific Polymerase Chain Reaction (MiMAS-PCR) directly from sputum specimens. From total 117 sputum samples, The MiMAS-PCR test demonstrated 98.4% and 100% specificity in MDR-TB and XDR-TB detection, respectively, but the sensitivity is only suitable for rapid detection of XDR-TB (100%), not for MDR-TB (43.9%). The MiMAS-PCR technique is suitable for the rapid detection of XDR-TB (in this study, only one sample), but not for MDR-TB from the sputum specimens. Other methods are required to identify MDR-TB rapidly for early treatment and to prevent spread to their surrounding contacts.

Evaluation of MALDI-TOF MS and an expanded custom reference spectra database for the identification and differentiation of Taylorella equigenitalis and Taylorella asinigenitalis

Misidentification between Taylorella equigenitalis, the causative agent of contagious equine metritis (CEM), and Taylorella asinigenitalis is observed by the gold standard culture method. The performance of matrix-assisted laser desorption ionization–time-of-flight mass spectrometry (MALDI-TOF MS) for Taylorella species identification was evaluated using 85 T. equigenitalis and 28 T. asinigenitalis strains selected on the basis of multilocus sequence typing data. Seven of the T. equigenitalis and 9 of the T. asinigenitalis strains were used to generate in-house reference spectra to expand the existing commercial Bruker database. Two bacterial incubation times and 3 different sample preparation procedures were compared. Overall, we demonstrated the usefulness of MALDI-TOF MS as a differential diagnostic tool for CEM; however, commercial spectra databases should be expanded with T. asinigenitalis reference spectra to achieve the expected performance. Moreover, direct spotting of 48-h colonies was not only the most efficient protocol but also the easiest to implement in a clinical setting.

Comparison of Xpert GBS v. culture for rapid detection of group B streptococcus in pregnant women: Sensitivity, specificity and predictive values.

Group B streptococcus (GBS) is a leading cause of invasive disease, particularly in newborns. Seventy-five percent of neonates will be colonised by mothers carrying the organism. Confirmation of maternal colonisation with GBS is essential for prompt treatment and prevention of neonatal sepsis. The current gold standard of culture for isolation of GBS has a disadvantage of long turnaround time (24 - 72hours). Rapid assays are required to determine maternal carriage of GBS. To determine the usefulness of the Xpert GBS technology v. culture methods to detect GBS carriage in pregnant women. This was a prospective observational study of 284 pregnant women between 26 and 37 weeks' gestation. Two vaginorectal swabs were collected from each participant. One swab was processed using the gold-standard culture method, while the second swab was processed using the Xpert GBS assay. The performance of the Xpert GBS assay was then compared with that of the culture method. Two swabs were processed from each of 284 pregnant women between 26 and 37 weeks' gestation. Culture detected 70 GBS isolates from a total of 279 specimens (25.1%), whereas the Xpert GBS detected 66 positive specimens (23.7%). The Xpert GBS assay had a sensitivity of 87% and specificity of 98%, with a positive predictive value of 92% and a negative predictive value of 96%. The Xpert GBS assay is a rapid and sensitive tool for prenatal detection of GBS. The assay should ideally be available in every labour ward, where women can be screened for GBS on arrival.