16S rRNA Polymerase Chain Reaction Research Articles

A Rare Case of Streptococcus cristatus Spondylodiscitis Identified by Bacterial 16S rRNA Polymerase Chain Reaction Sequencing: A Case Report and a Review of the Literature

A Rare Case of Streptococcus cristatus Spondylodiscitis Identified by Bacterial 16S rRNA Polymerase Chain Reaction Sequencing: A Case Report and a Review of the Literature

Relationship, association and distribution of Helicobacter pylori infection in endoscopy patients at a Tertiary Hospital in Makurdi, Benue State, Nigeria to the symptoms of nausea and vomiting

The distribution of H. pylori obtained from patients referred for endoscopy at Benue State University Teaching Hospital (BSUTH) Makurdi, Benue State, Nigeria in relation to nausea and vomiting was examined in this study. To isolate H. pylori, gastric biopsy samples were obtained from the antrum of 80 consenting patients, and questionnaires were administered to them. The biopsies were placed in Brain Heart Infusion (BHI) broth and were used for isolation. Genomic Deoxyribonucleic Acid (gDNA) was extracted from the biopsies using ReliaPrep spin column method and used for singleplex Polymerase Chain Reaction (PCR). Out of the 80 patients, 24 (30%) had H. pylori infection as detected by 16S rRNA PCR. There was no significant association between infection and nausea/vomiting (OR = 5.622, 95% CI: 0.683-46.274; p = 0.076), or infection and hematemesis, common symptoms reported by patients (OR = 0.911, 95% CI: 0.304 - 2.729; p = 0.870). The research sheds light on the infection, prevalence and distribution of H. pylori in endoscopy referral patients at BSUTH, with respect to nausea and vomiting, and provides valuable information for both clinicians and researchers.

Lumbar Osteomyelitis and Epidural Abscess Caused by Cardiobacterium hominis—First Reported Case in the Pediatric Population: The Role of Universal 16S rRNA Gene PCR and Sequencing

Abstract This report presents a case of a 14-year-old female patient diagnosed with lumbar (L2–L3) epidural abscess and vertebral osteomyelitis caused by Cardiobacterium hominis. The diagnosis was made using universal 16S rRNA polymerase chain reaction. To the best of our knowledge, this is the first reported case of C. hominis lumbar epidural abscess and vertebral osteomyelitis in pediatric population. This case serves to illustrate the potential value of polymerase chain reaction and DNA sequencing in diagnosing culture-negative vertebral osteomyelitis and the potential role of C. hominis in causing this infection.

Extended-Spectrum Beta-Lactamases Genes in Klebsiella pneumoniae Isolates Obtained from Patients in Intensive Care Units

Background: Drug-resistant hospital-acquired infections (HAIs) are a growing concern in modern medicine throughout the world. Klebsiella pneumoniae is one of the most prominent causative agents of multidrug-resistant nosocomial infections. It is also widely recognized for having a high resistance level to many antibiotic classes, particularly beta-lactams. Carbapenemase-producing K. pneumoniae has been identified as a major global cause of HAIs with adverse clinical outcomes. Therefore, it is of the utmost importance to have an in-depth understanding of the antimicrobial resistance (AMR) genetic determinants of this bacterium to stop the spread of highly resistant K. pneumoniae in healthcare facilities and the resulting patient morbidity and mortality. Objectives: This study aimed to investigate the AMR pattern of K. pneumoniae isolates obtained from intensive care units (ICUs), with a focus on extended-spectrum beta-lactamases (ESBLs) genes blaCTX-M, blaGES, and blaIMP. Methods: A total of 105 K. pneumoniae isolates obtained from the sputum samples of ICU patients were identified and confirmed using standard microbiological tests and 16S rRNA polymerase chain reaction (PCR). The antibiotic susceptibility test was performed for all the isolates. The presence of ESBL genes was determined phenotypically and by PCR. Results: The highest level of resistance was observed against ceftazidime (100%), cefotaxime (99%), and imipenem (93.3%). Approximately 87.6% and 39% of the isolates were sensitive to colistin and gentamicin, respectively. Phenotypic ESBL production was observed in 16 isolates, and the prevalence of blaCTX-M was 86.7%. No blaGES and blaIMP genes were detected. Conclusions: Periodic investigation of AMR-mediating genes is essential due to the high prevalence of ESBL genes in HAIs. The presence of other ESBL genes needs to be investigated for a more accurate understanding of the AMR status of K. pneumoniae in healthcare settings.

Feasibility and efficacy of newly developed eco-friendly, automatic washer for endoscope using electrolyzed alkaline and acidic water.

As well as preventing nosocomial and healthcare-associated infections, a reliable and eco-friendly washer for medical equipment would also be safe for the global environment. The aim of this study was to evaluate the efficacy of a newly developed automatic washing system (Nano-washer) that uses electrolyzed water and ultrasonication without detergent for washing endoscopes. Patients who underwent laparoscopic lobectomy or laparoscopic colectomy at Nagasaki University between 2018 and 2022 were included. A total of 60 cases of endoscope use were collected and classified according to endoscope washing method into the Nano-washer group (using no detergent) (n = 40) and the manual washing group (n = 20). Protein and bacterial residues were measured before and after washing, using absorbance spectrometry and 16S rRNA polymerase chain reaction. The effectiveness of protein and bacterial removal and endoscope surface damage after washing were compared under specular vision between the groups. Nano-washer did not use detergent unlike manual washing. There was no difference in demographic or clinical characteristics between the groups except for the presence of comorbidities in the lobectomy group (Nano-washer, 85%; manual washing, 40%, P = .031). Compared with the manual washing group, residual protein levels in the Nano-washer group were significantly reduced after washing (lobectomy, 0.956 mg/mL vs 0.016 mg/mL, P < .001; colectomy, 0.144 mg/mL vs 0.002 mg/mL, P = .008). Nano-washer group showed a significant reduction in bacteria between before and after lobectomy (9437 copies/cm2 vs 4612 copies/cm2 , P = .024). Nano-washer is a promising, effective, and eco-friendly automatic washing device that is safer and more efficient than manual washing.

Antimicrobial susceptibility patterns and molecular phylogenetics of Proteus mirabilis isolated from domestic rats: An environmental driver to antimicrobial resistance in public health in Arusha Tanzania

Proteus mirabilis (P. mirabilis) is a bacterial pathogen contributing to opportunistic infections, nosocomial outbreaks, and mostly hematogenous ascending urinary tract infections. It has repeatedly been found in rats. Due to rat-human interaction, rats are likely responsible for spreading these bacteria and their antimicrobial-resistant. This study was performed to genetically characterize and assess antimicrobial susceptibility patterns of P. mirabilis isolated from rats cohabiting with humans in Arusha municipality, Tanzania. A total of 139 rats were trapped from March to May 2021 and identified at the species level using morphological and morphometric features. Deep-intestinal swabs were obtained and pre-enriched in buffered peptone water. P. mirabilis was isolated by conventional culture and biochemical methods and confirmed by 16S rRNA polymerase chain reaction and sequencing. Phylogenetics was used to assess the similarities of the isolates. Antimicrobial susceptibility test was done by disk diffusion method using seven antibiotics, including tetracycline, ciprofloxacin, gentamicin, cefotaxime, trimethoprim-sulfamethoxazole, azithromycin, and ampicillin. Resistance genes blaTEM, tetA, tetB, mphA, blaSHV, blaCTX-M, sul1, and sul2 were traced in each isolate using PCR. Mixed rat species, Rattus rattus (55.4%), Mus musculus (15.8%), and Mastomys natalensis (28.8%), were captured. P. mirabilis was isolated from four (2.9%) Rattus rattus samples. By PCR and sequencing, all were confirmed as P. mirabilis and 100% similar to strains from GenBank. Three isolates showed multidrug resistance (MDR) against trimethoprim-sulfamethoxazole, azithromycin, and ampicillin, while all isolates were resistant to azithromycin and ampicillin, and susceptible to ciprofloxacin, gentamicin, and cefotaxime. Three were resistance to trimethoprim-sulfamethoxazole and intermediate to tetracycline. PCR analysis detected tetA, blaTEM, sul1, and sul2 resistance genes. Constructed phylogenetic tree showed that all isolates from this study were closely related to isolates from Tunisia. The study has discovered the first P. mirabilis isolates from rats in Tanzania with antimicrobial resistance traits that could be of public health concern.

Parapneumonic effusion in children: Rapid pathogen detection in pleural fluid using multiplex bacterial PCR.

Parapneumonic effusion in children: Rapid pathogen detection in pleural fluid using multiplex bacterial PCR.

1328. Paenibacillosis: An Emerging Cause of Neonatal Sepsis and Postinfectious Hydrocephalus

Abstract Background The etiology of neonatal sepsis is often not identified. Molecular methods can identify pathogens that culture-based methods miss. Most cases of neonatal sepsis globally are treated empirically per WHO guidelines with intravenous ampicillin and gentamicin, which may not be the best regimen for all pathogens. Methods We prospectively enrolled 800 neonates presenting with signs of sepsis to two Ugandan hospitals. Blood and cerebrospinal fluid were subjected to 16S rRNA sequencing, which identified Paenibacillus thiaminolyticus in 33/800 (4%) neonates. We confirmed the presence of P. thiaminolyticus by quantitative polymerase chain reaction (PCR). We describe neonatal and birth characteristics, presenting signs, and 12-month developmental outcomes for neonates with paenibacillosis. We performed antibiotic susceptibility testing and genomic analyses on three clinical isolates successfully grown in the laboratory. Results Neonates presented at a median age of 3 (1, 7) days. Fever (86%), irritability (78%) and seizures (52%) were common presenting signs (Figure). Most neonates were born vaginally (73%) at a medical facility (79%). Twelve (36%) had an adverse outcome: 5 (15%) neonates died; 4 (14%) survivors developed postinfectious hydrocephalus and three (9%) additional survivors had neurodevelopmental impairment. All three isolates were resistant to vancomycin, two were resistant to penicillin and ampicillin and one was unlikely to be sensitive to ceftriaxone; all were susceptible to gentamicin and meropenem. The genomes of all three strains contained multiple beta-lactamase genes and a cluster of genes that encodes a type IV pilus. Clinical signs at presentation for neonates with good and poor outcomes followng paenibacillosis Conclusion Molecular methods such as 16S rRNA sequencing and PCR can be used to improve the identification of pathogens causing neonatal sepsis. Paenibacillosis is an important emerging cause of neonatal sepsis in Uganda and is likely an underrecognized cause of postinfectious hydrocephalus in the region and possibly elsewhere. Antibiotics commonly used for neonatal sepsis may be inadequate for the treatment of paenibacillosis. Additional studies to understand the pathophysiology and optimal treatment of this novel infection are urgently needed to prevent neonatal mortality and morbidity including postinfectious hydrocephalus. Disclosures Jessica E. Ericson, MD, MPH, Abbvie: Advisor/Consultant Abhaya Kulkarni, MD, MSc, PhD, Medtronic: Advisor/Consultant.

Molecular study of Enterobacter cloacae isolated from leukemia patients

Introduction and Aim: Polymerase chain reaction (PCR) of 16S rRNA and virulence genes associated with Type Three Secretion System (TTSS) has been used as a rapid method for the identification of the pathogen Enterobacter cloacae in leukemia patients. Rapid diagnosis of this pathogen becomes necessary for starting a proper treatment in these patients. In the current study, we aimed to isolate Enterobacter cloacae from leukemia patients and study the TSSS genes associated with these isolates using molecular methods. Materials and Methods: E. cloacae isolates identified using biochemical tests was molecular confirmed by 16S rRNA polymerase chain reaction (PCR). Genomic DNA extracted was also subjected to the Type Three Secretion System (TTSS) associated virulence genes escV and ascV using specific primers. Results: 30 (23.07%) out of the 130 blood samples tested in this study were identified as E. cloaca by biochemical tests. Further confirmation using molecular methods showed only 11 of these isolates to be 16S rRNA positive. Few of these isolates were positive for the TTSS associated ASCV gene of the samples positive for presence of the ascV gene. All strains were negative for the escV gene. Conclusion: PCR is the best technique in comparison with other conventional methods for the diagnosis of E. cloacae in leukemia patients because of its safety, high sensitivity, specificity, and speed.

Identification of Streptomyces spp. in a Clinical Sample: Always Contamination? Results of a French Retrospective Study.

Background Streptomyces are environmental gram-positive bacilli that can cause ubiquitous mycetoma and, more rarely, invasive infections. We describe the clinical relevance of Streptomyces spp. identified in human samples and characteristics of patients with invasive Streptomyces infections.MethodsWe conducted a retrospective (2006–2017) study of Streptomyces isolates identified in clinical samples in French microbiology laboratories. Streptomyces genus was confirmed by a specific 16S rRNA polymerase chain reaction, and antibiotic susceptibility testing was performed by disk diffusion and trimethoprim-sulfamethoxazole minimum inhibitory concentration (E-test) if resistance was suspected. Patient characteristics, treatments, and outcomes were collected. Invasive infection was defined as a positive culture from a sterile site with signs of infection but without cutaneous inoculation.ResultsOf 137 Streptomyces isolates, all were susceptible to amikacin (113/113) and linezolid (112/112), and 92.9% to imipenem (105/113). Using disk diffusion, 50.9% (57/112) of isolates were susceptible to trimethoprim-sulfamethoxazole, but most of the apparently resistant isolates (25/36, 69.4%) tested by E-test were ultimately classified as susceptible. Clinical data were obtained for 63/137 (45.9%) isolates: 30 (47.6%) invasive infections, 8 (12.7%) primary cutaneous infections, 22 (34.9%) contaminations, 3 (4.7%) respiratory colonization. Patients with invasive infection were more frequently receiving corticosteroids than patients without invasive infection (11/30, 36.7%, vs 2/25, 8.0%; P = .03), and at 6-month follow-up, 14 of them were cured, 3 had relapsed, 4 were dead, and 9 were lost to follow-up.ConclusionsHalf of the clinical samples that grew Streptomyces were from patients with invasive infection. In that case, antimicrobial therapy should include 1 or 2 antibiotics among linezolid, amikacin, or imipenem.

Assessing Asymptomatic Malaria Carriage of Plasmodium falciparum and Non-falciparum Species in Children Resident in Nkolbisson, Yaoundé, Cameroon.

Malaria is still a threat to public health as it remains the first endemic disease in the world. It is a pervasive parasitic disease in tropical and subtropical regions where asymptomatic malaria infection among humans serves as a significant reservoir for transmission. A rapid and correct diagnosis is considered to be an important strategy in the control of the disease especially in children, who are the most vulnerable group. This study assessed the prevalence of asymptomatic malaria in children at the Nkolbisson health area in Yaoundé, Cameroon. A cross-sectional study design and a convenience sampling plan were used. A total of 127 participants were recruited after informed and signed consent from parents and/or guardians. Blood samples were collected by finger-pricking and venipuncture from children aged 6 months to 10 years and then screened for asymptomatic parasitemia by a rapid diagnostic test (RDT), light microscopy (LM) staining with Giemsa and 18S rRNA polymerase chain reaction (PCR) for speciation. The data were analyzed using SPSS version 20 software. The study identified 85 children who were positive from the PCR, 95 positive from the RDT and 71 from the LM, revealing a malaria prevalence of 66.9%, 74.8% and 55.9%, respectively. The prevalence was not observed to be dependent on the sex and age group of the participants. Plasmodium falciparum was the predominant species followed by Plasmodium malariae and then Plasmodium ovale. The RDT and LM had the same sensitivity (90.6%) with a slight difference in their specificity (RDT: 57.1%; LM: 54.8%). The RDT also demonstrated higher positive and negative predictive values compared with those of the LM.

Detection of bacterial DNA in synovial fluid in dogs with arthritis: a comparison between bacterial culture and 16S rRNA polymerase chain reaction

BackgroundSeptic arthritis (SA) is a serious condition in dogs that requires a prompt diagnosis and treatment to minimize long-term joint pathology. Although bacterial detection in synovial fluid (SF) through culture or cytology is often performed to confirm diagnosis, the sensitivity of these tests is low. The need for a reliable diagnostic tool to confirm the presence of bacteria in SF in humans has led to the increased use of 16S rRNA (i.e., ribosomal RNA) gene sequencing by polymerase chain reaction (16S rRNA PCR). The aim of this prospective clinical study was to compare the sensitivity and specificity of 16S rRNA PCR with bacterial culture on blood agar plates after pre-incubation of SF in paediatric blood bacterial culture bottles to identify bacteria in dogs with clinical signs of SA and to investigate the usefulness of these methods as diagnostic tools.ResultsTen dogs with clinical signs of SA, nine with osteoarthritis (OA, control group) and nine with clinical signs of immune-mediated polyarthritis (IMPA, second control group) were examined. Bacterial culture was positive in seven of 10 dogs with clinical SA, of which only two were positive by 16S rRNA PCR. The sensitivity of 16S rRNA PCR and bacterial culture analysis for dogs with clinical SA were 20% and 70%, respectively. All SF samples collected from control group (n = 9) and second control group (n = 14) animals were negative on culture, and 16S rRNA PCR rendered a specificity of 100%.ConclusionsOur study showed a lower sensitivity of 16S rRNA PCR than bacterial culture for dogs with clinical SA. Our findings suggest that there is currently no advantage in using 16S rRNA PCR as a diagnostic tool for dogs with clinical SA. Furthermore, our study indicates that pre-incubation in paediatric blood bacterial culture bottles before bacterial cultivation on blood agar plates might enhance bacterial culture sensitivity compared to other culture methods.

The Accuracy of 16S rRNA Polymerase Chain Reaction for the Diagnosis of Neonatal Sepsis: A Meta-Analysis.

Objective To determine the accuracy of 16S rRNA polymerase chain reaction (PCR) for the diagnosis of neonatal sepsis through a systematic review and meta-analysis. Methods Studies involving 16S rRNA PCR tests for the diagnosis of neonatal sepsis were searched in the PubMed, Medline, Embase, and Cochrane Library databases. The methodological quality of the identified studies was evaluated using the Quality Assessment of Diagnostic Accuracy Studies-2 (QUADAS-2), and the sensitivity, the specificity, the positive likelihood ratio (PLR), the negative likelihood ratio (NLR), the diagnostic odds ratio (DOR), and the area under the curve (AUC) of operator characteristic (SROC) curves were determined. Heterogeneity between studies was analyzed by metaregression. Stata 14.0 and Meta-disc 1.4 software were used for the analyses. Results This meta-analysis included 19 related studies. The analysis found a sensitivity of 0.98 (95% CI: 0.85-1), specificity of 0.94 (95% CI: 0.87-0.97), PLR of 16.0 (95% CI: 7.6-33.9), NLR of 0.02 (95% CI: 0.00-0.18), DOR of 674 (95% CI: 89-5100), and AUC of 0.99 (95% CI: 0.97-0.99). Metaregression analysis identified Asian countries, arterial blood in blood samples, and sample size > 200 as the main sources of heterogeneity. This meta-analysis did not uncover publication bias. Sensitivity analysis showed that the study was robust. Fagan's nomogram results showed clinical usability. Conclusions The results from this meta-analysis indicate that 16S rRNA PCR testing is effective for the rapid diagnosis of neonatal sepsis.

Molecular Identification and Antimicrobial Sensitivity Profile of Bacteria Isolated from Food Handlers in Akure Nigeria

Hand washing and hand swab samples culminating to a total of 477 samples were collected from different food vending locations in Akure. Bacteria were isolated and identified, after which the sensitivity patterns of the isolates to standard antibiotics and plant extracts were determined using disc diffusion agar and well diffusion methods, respectively. The bacterial isolates include Escherichia coli, Klebsiella oxytoca, Klebsiella pneumoniae, Micrococcus luteus, Proteus mirabilis, Proteusvulgaris, Pseudomonas aeruginosa, Salmonella enteritica, Salmonella typhi, Serratia marcescens, Shigella sonnei, Staphylococcus epidermidis, Staphylococcus aureus and, Streptococcus pyogenes. Staphylococcus aureus was observed to be the most prominent bacteria harboured by the food handlers. The isolated bacteria were subjected to 16s rRNA polymerase chain reaction, DNA gene extraction and sequence blasting using the amplified length of 1500 base pair. The sequence obtained was blasted in National Centre for Biotechnology Information (NCBI) database. Based on the 16s rRNA sequences, the following bacteria were confirmed; Escherichia coli str. K12 substr. DH10B, Klebsiella pneumoniae strain J1, Proteus mirabilis strain HI4320, Pseudomonas fluorescens SBW25, Salmonella enterica subsp.enterica serovar Typhi str. CT18, Salmonella enterica subsp. enterica serovar Infantis, Shigella flexneri 2a str. 2457T and Staphylococcus cohnii subsp. cohnii strain 532 Contig16. Phylogeny was employed in the phylogenetic tree construction due to its robustness, reliability and less-laboriousness. The result of the sequence was aligned and the phylogenetic tree constructed shows the evolutionary relationship between eight bacterial species. Two clusters were generated; Salmonella enterica subsp. enterica serovar Typhi, Salmoenella typhi LN854584.1 Staphylococcus cohnii, and Staphylococcus aureus are in cluster 1, Proteusmirabilis NC0100554.1, Bacillus thuringiensis KY652120.1 and Bacillus subtilis MG231261.1 is incluster 2. However, bacteria in the same cluster were observed to have different phenotypic characteristics. The antibacterial efficacy of the selected medicinal plant extracts (Ocimum gratissimum and Annona muricata) were copared to that of standard antibiotics (Ciprofloxacin, Nitrofurantoin, Ampliclox, and Chloramphenicol) using three different solvents (methanol, water and petroleum ether) at 50 mg/ml, 100 mg/ml, 200mg/ml and 300mg/ml, concentrations. Bioactive characterization of the selected plants revealed the presence ofvarious active components such as Thymol, Eugenol, Caryophyllene, Gamma-terpinene, 8-methyl-4-vanillyl-7-nonenamide, N-4-Hydroxy-3-methoxybenyl 8methyldec 6 enamide, DL-6, 8-tio acidamide and N-4-Hydroxy-3-methoxyphenyl methyl nonanamide. Phytochemical analysis of the plants affirmed the presence of Tannins, Alkaloids, Flavonoids, Anthraquinone and Saponins in O. gratissimum and A. muricata. It can be deduced from this study that food handlers play a major role in the transfer of bacterial contaminants to the edible food. Also, the metabolites present in O. gratissimum and A. muricata render these plants cost effective alternative source of therapy to food borne illnesses.

A case of infective endocarditis caused by Arthrobacter woluwensis

Corynebacteria are rare causative agents of infective endocarditis. This is a reported case of a destructive aorto-mitral infective endocarditis caused by Arthrobacter woluwensis. Microbial identification was achieved by 16S rRNA polymerase chain reaction on valve tissue samples. Outcome was favorable after surgical valve replacement and 4-week antibiotic treatment.

A Cross-Sectional Study on Molecular Detection of Helicobacter pylori cytotoxin-associated gene A and 16SrRNA Gene from Gastric Biopsy Specimens.

Introduction:The aim of the study is relative proportion of cytotoxin-associated gene A (cagA) virulence marker in Helicobacter pylori isolates and gastric biopsy samples by polymerase chain reaction (PCR).Methods:This cross-sectional study was conducted at a tertiary care hospital setting. Gastric biopsy tissues from 200 patients, suffering from upper gastrointestinal tract disorders, were examined for H. pylori infection using methods, such as hematoxylin and eosin (H and E) staining, 16S rRNA (Ribosomal ribonucleic acid), and cagA gene PCR. Chi-square and kappa statistics were used to find the association and agreement between the tests, respectively; P ≤ 0.05 was considered statistically significant. Screening tests' accuracy was calculated in terms of sensitivity and specificity along with positive and negative predictive values.Results:Out of 200 patients, H. pylori was detected in 14.5%, 48.5%, and 31% patients by H and E staining, 16S rRNA, and cagA PCR, respectively. Sensitivity and specificity of cagA PCR as compared to H and E staining were 89.6% and 78.9%, respectively.Conclusions:CagA detection directly from biopsy specimen by PCR can potentially and rapidly determine the patient's status, especially when at a higher risk of peptic ulcer.

Rapid and Accurate Species-Specific PCR for the Identification of Lethal Chironex Box Jellyfish in Thailand.

Box jellyfish are extremely potent venom-producing marine organisms. While they have been found worldwide, the highest health burden has been anticipated to be the tropical Indo-Pacific of Southeast Asia (SEA). At least 12 Cubozoan species have now been documented in Thai waters, and many of them inflict acutely lethal strings, especially those under the order Chirodropida. Our previous study has successfully differentiated species of box jellyfish using DNA sequencing to support the morphological study. In this study, we specifically designed polymerase chain reaction (PCR) primers for the 16S ribosomal RNA (rRNA) gene and the mitochondrial DNA cytochrome oxidase subunit I (COI) gene of lethal Thai Chironex species. The SYBR green-based real-time PCR panel was performed for rapid species identification. The sensitivity and specificity of the panel were determined by testing samples of different species. Moreover, we applied the panel to the tentacle sample from a real patient, which helped confirm the animal-of-cause of envenomation. Our results show a success for species identification of box jellyfish using 16S rRNA and COI PCR panel, which revealed congruence between molecular and morphological identification. Furthermore, the panel worked very well with the unknown samples and jellyfish tissue from the real envenomation case. The results demonstrated that molecular panels were able to identify three species of Chironex box jellyfish both rapidly and accurately, and can be performed without having a complete specimen or morphological study.

Continuous Cell Lines from the European Biting Midge Culicoides nubeculosus (Meigen, 1830).

Culicoides biting midges (Diptera: Ceratopogonidae) transmit arboviruses of veterinary or medical importance, including bluetongue virus (BTV) and Schmallenberg virus, as well as causing severe irritation to livestock and humans. Arthropod cell lines are essential laboratory research tools for the isolation and propagation of vector-borne pathogens and the investigation of host-vector-pathogen interactions. Here we report the establishment of two continuous cell lines, CNE/LULS44 and CNE/LULS47, from embryos of Culicoides nubeculosus, a midge distributed throughout the Western Palearctic region. Species origin of the cultured cells was confirmed by polymerase chain reaction (PCR) amplification and sequencing of a fragment of the cytochrome oxidase 1 gene, and the absence of bacterial contamination was confirmed by bacterial 16S rRNA PCR. Both lines have been successfully cryopreserved and resuscitated. The majority of cells examined in both lines had the expected diploid chromosome number of 2n = 6. Transmission electron microscopy of CNE/LULS44 cells revealed the presence of large mitochondria within cells of a diverse population, while arrays of virus-like particles were not seen. CNE/LULS44 cells supported replication of a strain of BTV serotype 1, but not of a strain of serotype 26 which is not known to be insect-transmitted. These new cell lines will expand the scope of research on Culicoides-borne pathogens.

Parity, exposure to cigarette smoke and the presence of bacteria Stenotrophomonas maltophilia are related to preterm labor incidence

This study aimed to determine the relationship of parity, exposure to cigarette smoke, and the presence of bacteria Stenotrophomonas maltophilia with the incidence of preterm labor. It was a cross-sectional study, the sampling was carried out using a purposive sampling technique for maternity women at the Siti Fatimah Makassar Mother and Child Hospital, Siti Khadija I Makassar Mother and Child Hospital, and Makassar City Regional General Hospital in June until August 2019. The study sample consisted of 50 post-partum mothers and their placenta, consisting of 25 preterm mothers and 25 term mothers. Criteria for respondents in this study were maternal pregnancies at gestational age>22-36 weeks for the case group, and ≥37 weeks for the control group while the exclusion criteria were mothers with hypertension, preeclampsia, placenta previa, placental abruption, and HIV. Respondent characteristics were collected using a checklist sheet. Bacteria on the placenta were detected using 16S rRNA Polymerase Chain Reaction (PCR). Characteristics of age, Body Mass Index (BMI), premature rupture of membrane History, abortion history, and preterm birth history did not have significant relationship with the incidence of preterm labor, while parity (OR=3.03, 95% CI: 1.2-12.0), cigarette smoke exposure (OR=4.03, 95% CI: 1.2-13.5), Stenotrophomonas maltophilia bacteria (OR=5.90, 95% CI: 1.11-31.20) significantly increases the risk of preterm labor. Parity, exposure to cigarette smoke, and the bacteria Stenotrophomonas maltophilia are considered as predisposing factors that are closely related to the incidence of preterm labor.

The Clinical Impact of 16S Ribosomal RNA Polymerase Chain Reaction Bacterial Sequencing in Infectious Endocarditis

Introduction Cases of possible and/or culture-negative endocarditis continue to be a diagnostic challenge. Performing bacterial 16S ribosomal RNA polymerase chain reaction (16S rRNA PCR) sequencing on cardiac valves allows providers to acquire microbiologic information that was previously unobtainable. However, few publications address how the polymerase chain reaction (PCR) results impact clinical management in endocarditis patients. Methods Between July 1, 2014, and December 31, 2018, we reviewed the results of all 16S rRNA PCR bacterial sequencing tests collected from cardiac valves at the University of Michigan. Each patient's chart was then evaluated by 2 independent infectious diseases physicians to determine whether the medical plans were impacted by the results. Results Forty-one patients were identified with associated 16S rRNA PCR testing from 165 explanted cardiac valves. Eighteen samples (43.9%) were positive, and 5 (12.2%) of 41 patients had their management plans changed based on the PCR findings. In 23 (56.1%) of 41 cases, the PCR result was never referenced by any medical provider in the electronic medical record. There were 7 cases (17.1%) where patients received 6 weeks of antibiotics despite presenting with possible culture-negative endocarditis, noninfectious operative findings, and negative valve PCRs, which were not reviewed. Conclusions 16S rRNA PCR sequencing is a useful tool for obtaining a microbiologic diagnosis in cases of possible or culture-negative endocarditis. The test has potential to impact patient care and in a subset of cases could be used to deescalate antibiotic therapy. However, testing delays and cumbersome resulting methods impede bacterial sequencing from reaching its full potential as a diagnostic modality.