Conventional Culture Methods Research Articles

The application of targeted nanopore sequencing for the identification of pathogens and resistance genes in lower respiratory tract infections.

Lower respiratory tract infections (LRTIs) are one of the causes of mortality among infectious diseases. Microbial cultures commonly used in clinical practice are time-consuming, have poor sensitivity to unculturable and polymicrobial patterns, and are inadequate to guide timely and accurate antibiotic therapy. We investigated the feasibility of targeted nanopore sequencing (TNPseq) for the identification of pathogen and antimicrobial resistance (AMR) genes across suspected patients with LRTIs. TNPseq is a novel approach, which was improved based on nanopore sequencing for the identification of bacterial and fungal infections of clinical relevance. This prospective study recruited 146 patients suspected of having LRTIs and with a median age of 61 years. The potential pathogens in these patients were detected by both TNPseq and the traditional culture workups. We compared the performance between the two methods among 146 LRTIs-related specimens. AMR genes were also detected by TNPseq to prompt the proper utilization of antibiotics. At least one pathogen was detected in 133 (91.1%) samples by TNPseq, but only 37 (25.3%) samples contained positive isolates among 146 cultured specimens. TNPseq possessed higher sensitivity than the conventional culture method (91.1 vs. 25.3%, P < 0.001) in identifying pathogens. It detected more samples with bacterial infections (P < 0.001) and mixed infections (P < 0.001) compared with the clinical culture tests. The most frequent AMR gene identified by TNPseq was bla TEM (n = 29), followed by bla SHV (n = 4), bla KPC (n = 2), bla CTX-M (n = 2), and mecA (n = 2). Furthermore, TNPseq discovered five possible multi-drug resistance specimens. TNPseq is efficient to identify pathogens early, thus assisting physicians to conduct timely and precise treatment for patients with suspected LRTIs.

Multiplex high-resolution melting assay for simultaneous detection of five key bacterial pathogens in urinary tract infections: A pilot study.

The diagnosis of urinary tract infections (UTIs) is usually based on the results of urine culture, but it is time-consuming, labor-intensive and has a low sensitivity. The aim of this study was to develop multiplex high-resolution melting assay (MHRM) for the simultaneous detection of five common bacterial pathogens (Escherichia coli, Klebsiella pneumoniae, Staphylococcus saprophyticus, Enterococcus faecalis, and group B streptococci (GBS)) directly from urine samples. A total of 287 urine specimens were evaluated by HRM assay and the results were compared with the conventional culture method. Five different melt curves generated and differentiated five bacterial pathogens. The detection limit of the MHRM assay was 1.5 × 103 CFU/ml for E. coli and K. pneumoniae and 1.5 × 102 CFU/ml for S. saprophyticus, E. faecalis and GBS. Compared to culture, the specificity of the MHRM assay ranged from 99.3 to 100%, and sensitivity 100% for all test pathogens. The MHRM assay developed in the current study might be functional tool for the diagnosis of UTIs and has the potential for direct detection of the organism in the clinical samples. Additionally, it creates results in less than 5 h, helping clinicians to start treatment with appropriate antimicrobial agents. This method could be a useful supplement to urine culture.

319. Clinical accuracy and utility of plasma microbial cell free DNA NGS for the diagnosis of invasive aspergillosis in patients with hematologic malignancy and COVID-19

Abstract Background Invasive aspergillosis (IA) is a great threat to the severely immunocompromised and patients with coronavirus disease (COVID-19). However, diagnosis of IA is often difficult due to need for invasive biopsy and low sensitivity of other diagnostic tests. Next-generation sequencing (NGS) of plasma cell free DNA (cfDNA) can be a novel non-invasive diagnostic modality. We evaluated the clinical accuracy and utility of microbial cfDNA NGS for the diagnosis of IA in patients with hematologic malignancy (HM) and COVID-19. Methods A single-center prospective study of plasma microbial cfDNA NGS was conducted in a tertiary-care hospital in South Korea. We enrolled adult patients with HM and COVID-19, who suspected IA and performed conventional diagnostic tests for IA. The results of NGS were compared with the diagnosis of IA through conventional methods. IA cases were diagnosed according to EORTC/MSG definitions in patients with HM, and modified AspICU criteria in patients with COVID-19. (Figure 1). Figure 1.Flow chart for the participant selection method used in this study Results Between March 2021 and January 2022, a total of 33 participants (22 [64.7%] male, median age 66.0 [50.5, 72.0]) were enrolled;19 participants with HM and 15 with COVID-19 were analyzed (Figure1 and Table1). In participants with HM, aspergillus cfDNA was detected in 100% of both proven (1/1) and probable (12/12) IA cases, and 33.3% of both possible (1/3) and no IA (1/3) cases. In participants with COVID-19, 46.2% of probable IA (6/13) showed positive aspergillus cfDNA. Detection rate of aspergillus cfDNA was significantly higher in proven/probable IA cases in participants with HM compared to participants with COVID-19. (100% vs 46.2%, p=0.005) (Figure 2). As shown in Table 2, among proven/probable IA cases, participants with positive aspergillus cfDNA showed significantly higher rate of having uncontrolled hematologic disease, receiving stem cell transplantation and recent chemotherapy. In three participants with HM, non-aspergillus strains confirmed by cfDNA NGS were in accordance with pathogens identified through conventional culture methods. Table 1.Baseline characteristics of participants suspected of invaisve aspergillosis performing microbial cell free DNA NGSFigure 2.cfDNA detection rate in participants with suspected fungal infection according to the EORTC/MSG or modified AspICU diagnostic criteria Conclusion Detection of aspergillus cfDNA showed high concordance in the results of conventional diagnostic methods in proven/probable IA of patients with HM and could be a helpful non-invasive approach to IA diagnosis in those populations. Disclosures All Authors: No reported disclosures.

329. Bedside Inoculation of Blood Culture Bottles Does Not Improve Ascites Culture Positivity Rate in Spontaneous Bacterial Peritonitis

Abstract Background Prior research demonstrates increased positivity rate for ascites cultures in spontaneous bacterial peritonitis (SBP) when directly inoculated into blood culture bottles at bedside, versus the “conventional method” - transportation of ascitic fluid in a sealed syringe to the laboratory, centrifuging, and plating on Chocolate, Blood, and MacConkey’s agars and in a Schaedler broth. We hypothesized that collection of ascites via direct inoculation into blood culture bottles would improve ascites culture positivity rate in SBP when compared to prior methods. Methods In November of 2021, our institution implemented a policy in which all ascites cultures were directly inoculated into blood culture bottles at bedside. We retrospectively reviewed all ascites cultures collected from May of 2021 through April of 2022, including all cultures with an absolute neutrophil count of greater than 250 cells/mm3. We excluded all samples from patients with secondary bacterial peritonitis and all samples collected in November. If a patient had multiple samples that met inclusion criteria, only the first was included for analysis. Our primary outcome was positivity rate of cultures pre- and post-implementation of this new policy. Results 1375 ascites cultures were collected. Of these, 52 met our inclusion criteria. These patients were on average 58 years old (range 33-74), 50% female, with a median MELD of 25.5 (range 8-40). The conventional method was positive in 7/37 (18.9%) cultures, and direct inoculation of blood cultures in 1/15 (6.67%), p = .27. Conclusion As opposed to the previous literature, we detected no difference in positivity rate between the conventional method of ascites culture compared to direct inoculation of blood cultures at the bedside. This difference from prior literature may be due to differences in time to inoculation, our smaller sample size, or overfilling of the bottles during inoculation. Disclosures All Authors: No reported disclosures.

Bioengineering Liver Organoids for Diseases Modelling and Transplantation.

Organoids as three-dimension (3D) cellular organizations partially mimic the physiological functions and micro-architecture of native tissues and organs, holding great potential for clinical applications. Advances in the identification of essential factors including physical cues and biochemical signals for controlling organoid development have contributed to the success of growing liver organoids from liver tissue and stem/progenitor cells. However, to recapitulate the physiological properties and the architecture of a native liver, one has to generate liver organoids that contain all the major liver cell types in correct proportions and relative 3D locations as found in a native liver. Recent advances in stem-cell-, biomaterial- and engineering-based approaches have been incorporated into conventional organoid culture methods to facilitate the development of a more sophisticated liver organoid culture resembling a near to native mini-liver in a dish. However, a comprehensive review on the recent advancement in the bioengineering liver organoid is still lacking. Here, we review the current liver organoid systems, focusing on the construction of the liver organoid system with various cell sources, the roles of growth factors for engineering liver organoids, as well as the recent advances in the bioengineering liver organoid disease models and their biomedical applications.

System of Rice Intensification in Indonesia: Research, Adoption and Opportunities

The conventional rice cultivation method is observed to be ineffective in increasing rice production in Indonesia, compounded further by the frequent occurrence of various diseases, pest infestations, and weather uncertainties. The long-term practice of using high agrochemical inputs has adversely affected natural resources such as water, soil, and air. Thus, farming transformation is much needed in order to address the nation’s food security. This transformation can be done via the adoption of agroecological practices which rely on biological processes rather than on agrochemicals to maintain soil fertility and protect plant health. The System of Rice Intensification (SRI) is an agroecological method of rice cultivation that relies primarily on creating conditions for healthy plant growth by minimizing inter-plant competition through individual planting and wide spacing, at the same time improving soil structure and functioning by applying organic amendments, facilitating soil-surface aeration during weeding, and managing water to avoid both continuous flooding and water-stressed conditions. This combination of management practices results in better rice growth and yield compared with standard cultivation methods. For this purpose, the impacts of the SRI method on the economic, environmental, and social perspectives were studied. We conclude that the high productivity obtained by the SRI farmers and field trials has proven the suitability of the SRI method for sustainable rice farming in Indonesia. SRI improves the productivity of land, water and increases rice yield by three times higher than the conventional method. SRI is now regarded as a good option to be practiced by farmers in order to bring about a new kind of green revolution that relies upon ecosystem services to increase yield.

Comparison of Three Phenotypic Methods of Carbapenemase Enzyme Detection to Identify Carbapenem-resistant Enterobacterales

The prevalence of multidrug-resistant gram-negative bacilli has increased worldwide. Critical care areas of most hospitals use carbapenem antibiotics for the empirical treatment of gram-negative bacterial (GNB) infections. In the last decade, there have been reports of the detection of carbapenem-resistant Enterobacterales (CRE). This rise in the spread of CRE presents a great challenge in the treatment of GNB infections and poses a serious threat to global health. To detect the burden of CRE and to characterize CRE, we used three phenotypic methods for the detection of carbapenemase enzymes. Using conventional aerobic bacterial culture methods, 150 Enterobacterales strains were isolated from various clinical samples. Identification of CRE was performed using multiple phenotypic detection methods, such as the Kirby Bauer disc diffusion method for meropenem (10 mcg) using the CLSI 2021 interpretation for meropenem, modified Hodge test (MHT), Carba NP test, and modified carbapenem inactivation method (mCIM) test. A total of 150 Enterobacterales strains were isolated over a period of 1 year. Among these, 66/150 (44%), 63/150 (43%), 64/150 (43%), and 65/150 (43%) were identified as CRE using the Kirby Bauer disc diffusion method, MHT, mCIM test, and Carba NP test, respectively. The sensitivity and specificity of MHT, mCIM, and Carba NP tests within 95% CI were 93.94%/100%, 96.97%/100%, and 98.48%/100%, respectively. The positive and negative predictive values of MHT, mCIM, and Carba NP tests were 100%/95.45%, 100%/97.67%, and 100%/98.82%, respectively. The accuracies of the MHT, mCIM, and Carba NP tests were 97.33%, 98.67%, and 99.33% respectively indicating a high burden of carbapenem resistance in Enterobacterales. Therefore, given the current statistics of carbapenem resistance, use of carbapenem as empiric treatment in the intensive care units of hospitals may not be beneficial. Identification of carbapenem resistance can help in the initiation of appropriate antimicrobial therapy. This study compares the accuracy and efficiency of Carba NP, mCIM, and MHT in detecting carbapenem-resistant Enterobacterales.

Optimization of 3D-aggregated spheroid model (3D-ASM) for selecting high efficacy drugs

Various three-dimensional (3D) cell culture methods have been developed to implement tumor models similar to in vivo. However, the conventional 3D cell culture method has limitations such as difficulty in using an extracellular matrix (ECM), low experimental reproducibility, complex 3D cell culture protocol, and difficulty in applying to high array plates such as 96- or 384-plates. Therefore, detailed protocols related to robust 3D-aggregated spheroid model (3D-ASM) production were optimized and proposed. A specially designed wet chamber was used to implement 3D-ASM using the hepatocellular carcinoma (HCC) cell lines, and the conditions were established for the icing step to aggregate the cells in one place and optimized ECM gelation step. Immunofluorescence (IF) staining is mainly used to simultaneously analyze drug efficacy and changes in drug-target biomarkers. By applying the IF staining method to the 3D-ASM model, confocal microscopy imaging and 3D deconvolution image analysis were also successfully performed. Through a comparative study of drug response with conventional 2D-high throughput screening (HTS), the 3D-HTS showed a more comprehensive range of drug efficacy analyses for HCC cell lines and enabled selective drug efficacy analysis for the FDA-approved drug sorafenib. This suggests that increased drug resistance under 3D-HTS conditions does not reduce the analytical discrimination of drug efficacy, also drug efficacy can be analyzed more selectively compared to the conventional 2D-HTS assay. Therefore, the 3D-HTS-based drug efficacy analysis method using an automated 3D-cell spotter/scanner, 384-pillar plate/wet chamber, and the proposed 3D-ASM fabrication protocol is a very suitable platform for analyzing target drug efficacy in HCC cells.

New Perspectives for Prosthetic Valve Endocarditis: Impact of Molecular Imaging by FISHseq Diagnostics.

The microbial etiology of prosthetic valve infective endocarditis (PVE) can be difficult to identify. Our aim was to investigate the benefit of molecular imaging technique fluorescence in situ hybridization (FISH) combined with 16S rRNA-gene polymerase chain reaction (PCR) and sequencing (FISHseq) for the analysis of infected prosthetic heart valves. We retrospectively evaluated the diagnostic outcome of 113 prosthetic valves from 105 patients with suspected PVE, treated in 2003-2013 in the Department of Cardiac Surgery, Charité University Medicine Berlin. Each prosthetic valve underwent cultural diagnostics and was routinely examined by FISH combined with 16S rRNA gene PCR and sequencing. We compared classical microbiological culture outcomes (blood and valve cultures) with FISHseq results and evaluated the diagnostic impact of the molecular imaging technique. Conventional microbiological diagnostic alone turned out to be insufficient, as 67% of preoperative blood cultures were noninformative (negative, inconclusive, or not obtained) and 67% of valve cultures remained negative. FISHseq improved the conventional cultural diagnostic methods in PVE in 30% of the cases and increased diagnostic accuracy. Of the valve culture-negative PVE cases, FISHseq succeeded in identifying the causative pathogen in 35%. FISHseq improves PVE diagnostics, complementing conventional cultural methods. In addition to species identification, FISH provides information about the severity of PVE and state of the pathogens (eg, stage of biofilm formation, activity, and localization on and within the prosthetic material). As a molecular imaging technique, FISHseq enables the unambiguous discrimination of skin flora as contaminant or infectious agent.

Recent Progress on Carotenoids Production from Microalgae: A review

The color of fruits and green vegetables is attributed to a pigment known as carotenoids. On the basis of presence and absence of oxygen, carotenoids are divided into two categories; carotenes which do not contain oxygen and xanthophylls which are oxygen carrier. Carotenoids are not only good anti-oxidant agents; they also exhibit anti-tumor properties. The structure of carotenoids consists of eight isoprene units, which forms a chain consisting of conjugated double bonds. The presence of conjugated double bond in carotenoids neutralize free radicals by accepting electrons from them. Till now, most of the carotenoids commercially available are produced chemically however, microalgae are a good source of carotenoids. Microalgae can produce the carotenoids in abundance using two-stage cultivation strategies. In first phase, microalgae are given with optimal growth conditions for maximum production of biomass, on the other hand, keeping the second phase for the storage of lipids or carotenoids in unfavorable conditions. The production of carotenoids in two-stage approach is increased by many times than the conventional single phase cultivation method. Carotenoids have many industrial applications.

Subaqueous free-standing 3D cell culture system for ultrafast cell compaction, mechano-inductive immune control, and improving therapeutic angiogenesis.

Conventional 3D cell culture methods require a comprehensive complement in labor-intensive and time-consuming processes along with in vivo circumstantial mimicking. Here, we describe a subaqueous free-standing 3D cell culture (FS) device that can induce the omnidirectional environment and generate ultrafast human adipose-derived stem cells (hADSCs) that efficiently aggregate with compaction using acoustic pressure. The cell culture conditions were optimized using the FS device and identified the underlying molecular mechanisms. Unique phenomena in cell aggregation have led to extraordinary cellular behavior that can upregulate cell compaction, mechanosensitive immune control, and therapeutic angiogenesis. Therefore, we designated the resulting cell aggregates as "pressuroid." Notably, external acoustic stimulation produced by the FS device affected the pressuroids. Furthermore, the pressuroids exhibited upregulation in mechanosensitive genes and proteins, PIEZO1/2. CyclinD1 and PCNA, which are strongly associated with cell adhesion and proliferation, were elevated by PIEZO1/2. In addition, we found that pressuroids significantly increase angiogenic paracrine factor secretion, promote cell adhesion molecule expression, and enhance M2 immune modulation of Thp1 cells. Altogether, we have concluded that our pressuroid would suggest a more effective therapy method for future cell therapy than the conventional one.

Phenotypic and genotypic profiling reveals a high prevalence of methicillin-resistant Staphylococcus aureus isolated from hospitals, houseflies and adjacent informal food retailers in Botswana.

The increasing occurrence of methicillin-resistant Staphylococcus aureus (MRSA) in the environment, food and healthcare systems is a global public health concern. MRSA is reported to cause food poisoning, osteomyelitis and pyogenic infections of the skin, and consequently has been categorized as a high-priority pathogen by the World Health Organization. Here, we determined the presence of MRSA in clinical (n=56), food (n=150) and housefly samples (n=970) collected from two hospitals in Botswana. Characterization based on phenotypic (antimicrobial resistance, biofilm production) and genotypic (antimicrobial resistance genes and integrons) profiles were performed on all isolates. Of the total samples tested, 64 were positive for MRSA following conventional culture methods and PCR amplification of the mecA and mecC genes for confirmation of presumptive MRSA isolates. The confirmed isolates included 71 % (95 % CI 83.2-59.6) from clinical, 9 % (95 % CI 14--4.8) from food, and 1 % (95 % CI 1.6-0.4) collected from housefly samples. In total 89 % (n=57) isolates in the current study showed a multidrug resistance phenotype, among these, resistance to β-lactams and glycoside antibiotic classes were predominant. Genotypic characterization showed the domination of the blatem gene (95 %) followed by fox (63 %) and tetO (19 %) whilst vanA was only reported in 13 % of the isolates. Integrons were detected in 50 % (32/64) of the total MRSA isolates, and we report a high prevalence of etd gene, detected in 67 % (43/64) of the isolates followed by eta 38 % (24/64) whilst tsst-1 (3%) was the least detected genetic determinant. The genes etb and PVL were not detected in a ll the tested MRSA isolates. We provide the first report on the prevalence of MRSA isolated from the clinical-food-vector nexus harbouring biofilm and blatem genes, and antibiotic resistance profiles in Botswana. These results are significant for risk-assessment analysis and the development of improved MRSA infection prevention and control strategies.

Assessment of Foodborne Bacterial Pathogens in Buffalo Raw Milk Using Polymerase Chain Reaction Based Assay.

Milk is a putrescible commodity that is extremely prone to microbial contamination. Primarily, milk and dairy products are believed to be easily contaminated by pathogenic microorganisms, including Listeria monocytogenes, Salmonella spp., and Staphylococcus aureus. The microbiological quality of raw milk and dairy products regarding foodborne pathogens is of paramount importance due to concern of human health. In this study 400 buffalo raw milk samples were screened for assessing the prevalence of L. monocytogenes, Salmonella spp., and S. aureus. This study implemented uniplex-polymerase chain reaction (u-PCR) and multiplex-polymerase chain reaction (m-PCR) assays for the fast simultaneous detection of these pathogens comparing to the conventional culturing methods. Raw milk samples were found contaminated with the prevalence of 2.2%, 4.0%, and 14.2% for L. monocytogenes, Salmonella spp., and S. aureus, respectively. These pathogens were detected with the optimized polymerase chain reaction assays after 6 h of enrichment. u-PCR and m-PCR demonstrated the limit of detection as 104, 102, and 10 cells/mL after 6, 12, 18, and 24 h for each culture of the pathogens. A high sensitivity (10 colony-forming unit [CFU]/mL) of the m-PCR protocol was noted. The developed protocol is a cost-effective and rapid method for the simultaneous detection of pathogens associated with raw milk and dairy industries.

PSI-4 Investigation of a Naturally Occurring XX Male Bull

Abstract The Y chromosome–linked gene sex-determining region (SRY) is believed to be the master initiator of male sex determination in almost all eutherian mammals, functioning to upregulate expression of its direct target gene SRY-related HMG box–containing gene 9 (SOX9). XX maleness in humans is a rare syndrome with a frequency of 1 in 20,000–25,000 males. Approximately 90% of these cases carry some amount of the Y chromosome sequences due to an illegitimate recombination between X and Y chromosomes, where most of these cases have normal male genitalia. A more severe manifestation, the remaining 10% do not have any Y-chromosome sequences, and typically have ambiguous genitalia. In this study, we report a case of an infertile bull. Physical examination of this purebred Holstein bull showed a phenotypically androgynous animal with a normal penis, cryptorchidism (2 diminutive testicles, only one descended), and with a skeletal conformation and stance usually associated with female individuals. Lymphocytes from heparin sodium anticoagulated blood were cultured, harvested, and prepared according to conventional cell culture methods revealed a normal 60, XX karyotype with no numerical or structural chromosomal aberrations. PCR analyses were negative for the SRY gene, and positive for androgen receptor gene on the X chromosome. No sperm were found in seminal fluid collected in a routine breeding soundness exam at 17 months of age. The bull was slaughtered at 18 months of age, and its reproductive tract was collected. The testicle weighed 18.9 g (descended), and 42.4 g (abdominal). Neither testicle/epididymal unit was appropriate size or fully developed. Two very enlarged vesicular glands (43.36 g and 52.52 g, normally each ~ 20 g) were observed alongside what appeared to be uterine tissue. Histology showed no tubular grooves in the ampulla and an epididymis devoid of sperm. The development of a male phenotype in SRY-negative XX individuals suggests that in addition to SRY, there are autosomal or X-linked genes involved in the sex-determining pathway. In humans, a dose change of the SOX9 gene, loss of function mutations in the WNT4, FoxL2, and RSPO1 testicular suppressing genes, and genomic rearrangements within the regulatory region of the SRY-box transcription factor 3 (SOX3) gene have all been associated with XX male sex reversal. To investigate whether a mutation or genomic rearrangement was responsible for the male phenotype in the SRY-negative 60, XX bull, DNA was collected from the bull and his sire and dam for long-read trio sequencing. These data may provide evidence for either a loss of function mutation in a gene normally inhibiting testes formation in XX individuals, or a gain of function mutation in a gene downstream of SRY in the sex determining pathway.

Trends in Asymptomatic Nasopharyngeal Streptococcus pneumoniae Carriage with qPCR and Culture Analysis.

We previously reported trends in pneumococcal nasopharyngeal carriage in the post-PCV13 era as detected by conventional culture methods. Our current aim is to assess if there are fundamental differences in the clinical and demographic features of children who have pneumococcal carriage detected by qPCR compared with culture analysis. The CASPER team conducted point-prevalence surveys in 2016 in healthy children in Calgary to determine trends in overall and serotype-specific pneumococcal nasopharyngeal carriage. Being 18 months of age (p = 0.009), having at least one sibling under 2 years of age (p = 0.04), having only sibling(s) over 2 years of age (p = 0.001), and childcare attendance (p = 0.005) were associated with carriage by qPCR methods only. Having only sibling(s) older than 2 years of age was associated with carriage detected by both qPCR and culture methods (p = 0.001). No clinical factors were associated with carriage detected by both qPCR and culture compared to qPCR methods only. Both analyses are suitable methods to detect carriage; however, qPCR analysis is more sensitive and more cost-effective. As there are no fundamental differences in the children that have pneumococcal nasopharyngeal carriage detectable by qPCR methods compared to conventional culture methods, molecular analysis may be a preferable option for future carriage studies.

Multiplex PCR: Aid to more-timely and directed therapeutic intervention for patients with infectious gastroenteritis.

Background:Multiplex PCR is a sensitive and rapid method compared with conventional methods. Therefore, we use multiplex PCR for the rapid detection of the four major intestinal pathogens causing gastroenteritis (Shigella spp., Campylobacter spp., Aeromonas spp. and Enterohemorrhagic Escherichia coli [EHEC]) in stool specimens.Materials and methods:A prospective randomized study using 200 stool samples obtained from patients presented with acute gastroenteritis during the study period (between February 2019 and December 2021). Bacteria in stool samples were identified using conventional culture methods and multiplex PCR for stool samplesResults:The identified organisms using conventional cultures; were Shigella (27%), Aeromonas species (10%) and EHEC (O157) (8%). Using multiplex PCR. Shigella spp. was the most commonly identified pathogen (detected in 40.5% of positive samples), followed by Aeromonas spp. (30%), EHEC (20%) and Campylobacter species was only detected in (1%) of positive samples. The diagnostic evaluation of multiplex PCR in relation to conventional method in diagnosis of Shigella, EHEC and Aeromonas showed, sensitivity of 100% (for each), specificity of 88.5%, 92.4%, 77.8% respectively. However, the diagnostic evaluation of multiplex PCR in relation to conventional method in diagnosis of Campylobacter showed specificity of 99% and NPV of 100%.Conclusions:Multiplex PCR is an accurate and rapid method for detection of common intestinal pathogens causing severe gastroenteritis. a rapid method that could be used in outbreaks for diagnosis of the common enteric pathogens causing fatal gastroenteritis.

Clinical diagnosis and etiology of patients with Chlamydia psittaci pneumonia based on metagenomic next-generation sequencing.

The incidence of severe Chlamydia psittaci (C. psittaci) pneumonia and coinfections is increasing. Early detection of this condition is needed to prevent negative outcomes, along with detailed descriptions of its associated clinical characteristics. Our study contributes by undertaking etiological analysis of patients with C. psittaci pneumonia based on metagenomic next-generation sequencing (mNGS). A retrospective analysis of 30 patients with C. psittaci pneumonia was undertaken and confirmed by mNGS or polymerase chain reaction (PCR). Clinical manifestations of the severe and non-severe C. psittaci pneumonia groups were compared for clinical reference. Etiological analyses were also performed to comprehensively understand pathogeny and coinfection with other respiratory pathogens in C. psittaci patients. The absolute value of lymphocytes (LYM) in the severe group was lower than in the non-severe group. At the same time, neutrophil-to-lymphocyte ratio (NLR), procalcitonin (PCT), alanine aminotransferase (ALT), D-II polymer, brain natriuretic peptide (BNP), myoglobin (MYO), and cardiac troponin I (cTnI) were significantly higher (P < 0.05) in the severe group. mNGS has a broader pathogen spectrum and can more sensitively detect C. psittaci and other low-abundance pathogens with a higher positive detection rate (100%, 13/13 vs. 46%, 6/13, P <0.05) than conventional culture methods. mNGS detected the following dominant species associated with C. psittaci in patients: bacteria (53.2%, 39% gram-positive, 61% gram-negative), fungi (12.9%), and viruses (33.9%). A total of 73.3% (11/15) of patients had suspected coinfections, with a coinfection rate of 91.7% (11/12) in the severe group. No coinfection or death occurred in the non-severe group. Prognosis in the severe group was poor, with a mortality rate of 27.3% (3/11) for patients with coinfection. Eight of 11 patients with coinfections (72.7%) recovered. In conclusion, the clinical symptoms of severe C. psittaci pneumonia manifested as abnormal inflammatory indicators, impaired liver function, myocardial injury, coagulation, and relatively low immune responses. The higher proportion of patients with coinfections in our study supports the use of mNGS for comprehensive early detection of respiratory infections in patients with C. psittaci pneumonia. Simultaneous early identification of coinfections would further improve the clinical treatment of these patients.

Molecular diagnostic of complicated pneumonia in the post-vaccine era.

The etiological diagnosis of community-acquired pneumonia (CAP) is still a challenge. We compared the conventional culture method and real-time polymerase chain reaction (RT-PCR) for the identification of Streptococcus pneumoniae in severe pediatric CAP. A retrospective hospital-based study was conducted. From 2012 to 2018, we have selected patients who had peripheral blood and/or pleural fluid collected for etiological investigation by RT-PCR. We included 113 children (median age: 3 years; interquartile range 1-6 years). RT-PCR increased the detection rate of S. pneumoniae by 6.5 times using blood samples and eight times using pleural fluid samples. Patients subjected to RT-PCR showed more prolonged hospitalization (p = 0.006), fewer comorbidities (p = 0.03), presence of pleural effusion (p = 0.001), presence of young forms of leukocytes (p = 0.001) and radiograph with characteristics of pneumonia (p = 0.002). The presence of pleural effusion [odds ratio (OR) = 14.7, 95% confidence interval (CI) 1.6-133.9; p = 0.01] and young forms of leukocytes (OR = 8.9, 95% CI 0.9-84.4; p = 0.05) were risk factors for positive RT-PCR pneumococcal when multivariate analysis was performed. RT-PCR is a reliable method for diagnosing severe CAP using sterile materials and a potentially applicable method in patients with clinical, radiological and non-specific laboratory characteristics of lower respiratory tract infection, especially in complicated cases with pleural effusion.

Synthesis and Evaluation of the Immunomagnetic Beads for Separation of the Salmonella typhimurium from Milk.

Salmonella causes zoonotic diseases in humans and many animal species. The bacteria could be spread through fecal-oral transmission and consumption of raw contaminated animal products. Despite the activities which are carried out for the prevention of salmonellosis, it causes economic losses. This study aimed to prepare immunomagnetic beads to separate the Salmonella bacteria from experimentally polluted milk samples. The antibodies were purified from the rabbit's hyperimmune sera and coupled to the Fe nanoparticles using diethylenetriaminepentaacetic acid (DTPA) as a linker. The synthesized particles were analyzed using electron microscopy. The limit of bacterial detection by using the immunomagnetic beads coupled with bacterial culture were tested in experimentally contaminated cow milk with Salmonella. The separated bacteria were identified by using bacterial culture and biochemical tests. Using immunomagnetic beads (IMB), the Salmonella bacteria were removed from milk samples, concentrated in sterilized PBS, and cultured in nutrient agar media. The conventional culture method detected the bacteria in samples polluted with at least 3×104 CFU/mL bacteria; however, isolated bacteria were separated from milk samples using IMB and defined on bacterial culture media. The 3 CFU/mL of S. Typhimuriumm were detected in experimentally polluted milk samples using the current immunomagnetic-culture method. The results suggested using the IMB-bacterial culture instead of the conventional culture method.

Conventional PCR versus Culture Method to Detect Common Fungal Pathogens in Patients with Respiratory Diseases

The study aimed to assess the frequency of invasive fungal infection in patients with respiratory diseases by conventional and molecular methods. This study included 117 Broncho alveolar lavage (BAL) samples were collected from patients with respiratory disease (79 male and 38 female) with ages ranged between (20-80) years, who attended Medicine Baghdad Teaching hospital and AL-Emamain AL-Khadhymian Medical City, during the period from September 2019 to April 2020. The results in PCR versus culture methods in this study showed that out of 117 samples of fungal infections 30(25.6 %) were detected by culture method, while the 24(20.5%) samples were detected by PCR technique, the most commonly diagnosed pathogenic fungi is Candida spp. followed by Aspergillus spp. By considering the culture method as a gold standard against the PCR technique, the results show that the sensitivity and specificity of PCR were (86.6%) and (100%) respectively.