rRNA PCR Assay Research Articles

Improving gonorrhoea molecular diagnostics: Genome mining-based identification of identical multi-repeat sequences (IMRS) in Neisseria gonorrhoeae

BackgroundCurable sexually transmitted infections (STIs), such as Neisseria gonorrhoeae (N. gonorrhoeae), are a major cause of poor pregnancy outcomes. The infection is often asymptomatic in pregnant women, and a syndrome-based approach of testing leads to a missed diagnosis. Culture followed by microscopy is inadequate and time-consuming. The gold standard nucleic acid amplification tests require advanced infrastructure settings, whereas point-of-care tests are limited to immunoassays with sensitivities and specificities insufficient to accurately diagnose asymptomatic cases. This necessitates the development and validation of assays that are fit for purpose. MethodsWe identified new diagnostic target biomarker regions for N. gonorrhoeae using an algorithm for genome mining of identical multi-repeat sequences (IMRS). These were then developed as DNA amplification primers to design better diagnostic assays. To test the primer pair, genomic DNA was 10-fold serially diluted (100 pg/μL to 1 × 10−3 pg/μL) and used as DNA template for PCR reactions. The gold standard PCR using 16S rRNA primers was also run as a comparative test, and both assay products were resolved on 1% agarose gel. ResultsOur newly developed N. gonorrhoeae IMRS-PCR assay had an analytical sensitivity of 6 fg/μL representing better sensitivity than the 16S rRNA PCR assay with an analytical sensitivity of 4.3096 pg/μL. The assay was also successfully validated using clinical urethral swab samples. We further advanced this technique by developing an isothermal IMRS, which was both reliable and sensitive for detecting cultured N. gonorrhoeae isolates at a concentration of 38 ng/μL. Combining isothermal IMRS with a low-cost lateral flow assay, we were able to detect N. gonorrhoeae amplicons at a starting concentration of 100 pg/μL. ConclusionTherefore, there is a potential to implement this concept within miniaturized, isothermal, microfluidic platforms, and laboratory-on-a-chip diagnostic devices for highly reliable point-of-care testing.

Detection of Staphylococcus Isolates and Their Antimicrobial Resistance Profiles and Virulence Genes from Subclinical Mastitis Cattle Milk Using MALDI-TOF MS, PCR and Sequencing in Free State Province, South Africa.

Staphylococcus species are amongst the bacteria that cause bovine mastitis worldwide, whereby they produce a wide range of protein toxins, virulence factors, and antimicrobial-resistant properties which are enhancing the pathogenicity of these organisms. This study aimed to detect Staphylococcus spp. from the milk of cattle with subclinical mastitis using MALDI-TOF MS and 16S rRNA PCR as well as screening for antimicrobial resistance (AMR) and virulence genes. Our results uncovered that from 166 sampled cows, only 33.13% had subclinical mastitis after initial screening, while the quarter-level prevalence was 54%. Of the 50 cultured bacterial isolates, MALDI-TOF MS and 16S rRNA PCR assay and sequencing identified S. aureus as the dominant bacteria by 76%. Furthermore, an AMR susceptibility test showed that 86% of the isolates were resistant to penicillin, followed by ciprofloxacin (80%) and cefoxitin (52%). Antimicrobial resistance and virulence genes showed that 16% of the isolates carried the mecA gene, while 52% of the isolates carried the Lg G-binding region gene, followed by coa (42%), spa (40%), hla (38%), and hlb (38%), whereas sea and bap genes were detected in 10% and 2% of the isolates, respectively. The occurrence of virulence factors and antimicrobial resistance profiles highlights the need for appropriate strategies to control the spread of these pathogens.

Presence of potentially novel Helicobacter pylori-like organisms in gastric samples from cats and dogs

While seven gastric non-Helicobacter pylori Helicobacter (NHPH) species are known to commonly colonize the stomach of cats and dogs, the potential of H. pylori and H. pylori-like organisms to infect animals remains controversial and was investigated in this study using gastric samples of 20 cats and 27 dogs. A Helicobacter genus-specific 16 S rRNA PCR assay, H. pylori-specific ureAB and glmM PCR assays and a nested PCR detecting 23 S rRNA in a Helicobacter genus-specific manner in a first round of PCR and a H. pylori-specific manner in a second round, were performed in combination with sequencing. Histopathological and anti-Helicobacter immunohistochemical evaluations were also performed. Based on 16 S rRNA sequence analysis, 39/47 animals (83%) appeared infected with canine/feline gastric NHPHs in the corpus and/or antrum. H. pylori-specific ureAB amplicons were obtained in samples of 22 stomachs (47%). One canine antrum sample positive in the ureAB assay was also positive in the H. pylori-specific glmM assay. While 36/47 (77%) animals had a positive sample in the first round of the nested 23 S rRNA PCR assay, all samples were negative in the second round. Sequence analysis of obtained amplicons and immunohistochemistry point towards the presence of unidentified H. pylori-like organisms in cats and dogs. Histopathological examination suggests a low pathogenic significance of the gastric Helicobacter spp. present in these animals. In conclusion, cats and dogs may be (co-)infected with gastric Helicobacter organisms other than the known gastric NHPHs. Culture and isolation should be performed to confirm this hypothesis.

18S rRNA Gene-Based Piroplasmid PCR: An Assay for Rapid and Precise Molecular Screening of Theileria and Babesia Species in Animals.

PurposeThe parasites of genera such as Babesia and Theileria are called piroplasmids due to the pear-shaped morphology of the multiplying parasite stages in the blood of the vertebrate host. Because of the enormous number of parasite species and the challenges of multiplex PCR, initial screening of samples using piroplasmid-specific PCR may be a more cost-effective and efficient technique to identify parasite species, especially during epidemiological studies. Accordingly, 18S rRNA PCR was standardized and optimized on common piroplasmids of different animals like cattle, buffaloes, sheep, goats, dogs, horses, and leopards.MethodsBloods samples from 1250 animals were collected from different animals in Junagadh district of Gujarat, India. 18S rRNA PCR was standardized and optimized as a primary method for molecular screening of piroplasms in domestic and wild animals. The method was checked for its analytical sensitivity and specificity. Parasite species-specific PCR and sequencing was used to validate the test. Moreover, in-silico restriction enzyme (RE) analysis was also done to assess its applicability in PCR–RFLP.ResultsPiroplasm infections were recorded in 63.3% of animals in Junagadh. The 18S rRNA PCR detected the piroplasmid DNA in as low as 39 picograms (pg) of whole blood genomic DNA isolated from microscopically Theileria positive blood samples and no reactivity was recorded from common but unrelated haemoparasites viz., Trypanosoma evansi, Hepatozoon spp., Anaplasma spp., and Ehrlichia canis was observed. The 18S rRNA PCR assay findings were confirmed by species-specific PCR and sequencing. Analysis of different sequences generated using 18S rRNA PCR revealed that the amplicon size of Babesia spp. is nearly 400 bp (393–408 bp) whereas Theileria spp. were more than 400 bp (418–424 bp). The percentage of sequence divergence among Babesia and Theileria spp. was 7.3–12.2% and 0.7–12.2%, respectively. In-silico restriction enzyme (RE) analysis reveals the presence of at least one site for a commercially available RE in 18S rRNA fragments of every parasite, which can differentiate it from its congeners.ConclusionsThe presented universal oligonucleotide-based PCR assay provides a highly sensitive, specific, cost-effective, and rapid diagnostic tool for the initial screening of piroplasmids infecting domestic and wild animals and is potentially helpful for large-scale epidemiological studies.Supplementary InformationThe online version contains supplementary material available at 10.1007/s11686-022-00625-2.

Investigation of the sequence profile of the Plasmodium falciparum 18SrRNA diagnostic target in isolates from naturally infected children with uncomplicated malaria

The gold standard for malaria diagnosis is evidence of parasitological confirmation but the traditional method by light microscopy and the routinely used rapid diagnostic tests (RDTs) have limitations. Molecular assays are known to have higher sensitivity and specificity but there are indications that they may also be compromised by genetic variability of the target sequence. The aim of this study therefore, was to evaluate the DNA sequence profile of the diagnostic target of the P. falciparum 18S rRNA PCR assay in field isolates from North-Central Nigeria. Blood samples were collected from 324 children presenting with acute febrile illness suspected to be uncomplicated malaria. Light microscopy and 18S rRNA PCR assay were employed to determine the presence of P. falciparum parasites. Sequence profile of the diagnostic target was evaluated by Sanger sequencing of the PCR products on ABI PRISM® 3100 DNA sequencer (PE Applied Biosystems). Of the 324 children enrolled into this study, 134 (41.4%) were positive for P. falciparum by microscopy while 218 (67.3%) were positive by PCR. The sensitivity of microscopy was 61.47%(95% CI= 57.88% - 69.64%) using the PCR assay as reference standard. The degree of agreement between microscopy and PCR as measured by Cohen's kappa was moderate (κ = 0.502, 95% CI = 0.463 - 0.715).Sequence analysis showed that the DNA target of the P. falciparum 18S rRNA PCR from the field isolates were highly conserved. Only one A>T single nucleotide polymorphism was found within the target sequence among the isolates in this study. This study showed that the DNA target sequence of the18S rRNA PCR assay is highly conserved in field isolates in the study region suggesting little or no impact of selective pressure acting on the locus and has implications for the enhanced sensitivity of the molecular assay.

Influence of low FODMAP-gluten free diet on gut microbiota alterations and symptom severity in Iranian patients with irritable bowel syndrome

Background and objectiveRecently, dietary restriction of fermentable carbohydrates (a low-FODMAP diet) in combination with a gluten-free diet (GFD) has been proposed to reduce the symptoms in irritable bowel syndrome (IBS) patients. Different studies reported that IBS has been associated with dysbiosis in the gut microbiota. Additionally, a few studies have reported inflammation in the gastrointestinal (GI) system of adults with IBS. In this study, we aimed to investigate the effects of low FODMAP-gluten free diet (LF-GFD) on clinical symptoms, intestinal microbiota diversity, and fecal calprotectin (FC) level in Iranian patients with IBS.DesignIn this clinical trial study, 42 patients with IBS (Rome IV criteria) underwent LF-GFD intervention for 6 weeks. Symptoms were assessed using the IBS symptom severity scoring (IBS-SSS), and fecal samples were collected at baseline and after intervention and analyzed by quantitative 16 S rRNA PCR assay. The diversity of gut microbiota compared before and after 6 weeks of dietary intervention. FC was also analyzed by the ELISA method.ResultsThirty patients (mean age 37.8 ± 10.7 years) completed the 6-week diet. The IBS-SSS was significantly (P = 0.001) reduced after LF-GFD intervention compared to the baseline. Significant microbial differences before and after intervention were noticed in fecal samples. A significant increase was found in Bacteroidetes, and the Firmicutes to Bacteroidetes (F/B) ratio was significantly (P = 0.001) decreased after the dietary intervention. The value of FC was significantly decreased after 6 weeks of dietary intervention (P = 0.001).ConclusionsOur study suggests that patients with IBS under an LF-GFD had a significant improvement in IBS symptoms severity, with reduced FC level following normalization of their gut microbiota composition. Further rigorous trials are needed to establish a long-term efficacy and safety of this dietary intervention for personalized nutrition in IBS.Clinical Trial Registry Number: IRCT20100524004010N26.

Molecular detection and characterization of novel haemotropic Mycoplasma in free-living mole rats from South Africa.

The importance of haemotropic Mycoplasma (haemoplasma) infections to animal and human health is increasingly recognised. Although wild rodents are known to host these bacteria, haemoplasma prevalence and diversity in small mammals is under-documented, globally. This is due to the reliance on molecular approaches to detect these unculturable, obligate bacteria and to a paucity of assays targeting informative gene regions. We attempted to address these challenges by evaluating the performance of three 16S rRNA PCR assays for detecting Mycoplasma in four African mole-rat species of the family Bathyergidae. This was achieved by screening DNA samples prepared from lung and liver samples of 260 bathyergids, sampled from natural and urban landscapes in the Western Cape Province with one published and two novel conventional PCR assays. Sequence-confirmed Mycoplasma presence guided calculations of the relative sensitivity and specificity of the assays and revealed that 26.5% of the rodents were haemoplasma-positive. Bathyergus suillus sampled near an informal human settlement had a significantly higher infection rate (42%) than the three bathyergid species sampled from natural settings, for which PCR-positivity ranged from 0% to 36%. The 16S rRNA gene phylogeny identified the presence of six Mycoplasma strains in bathyergids that form a novel monophyletic lineage belonging to the haemofelis group, with 16S rRNA and Rnase P gene phylogenies indicating that the bathyergid-associated haemoplasmas were novel and closely related to Mycoplasma coccoides. Assay sensitivity ranged from 60.3% to 76.8% and specificity from 94.8% to 100% and both were highest for the novel assay targeting a~300bp region of the 16S rRNA gene. Results confirm the presence of novel haemoplasma strains in bathyergid species from South Africa and emphasise the need for expanded studies on haemoplama prevalence, diversity, and transmission routes in other small mammal species from this biodiverse region.

Oral, Cloacal, and Hemipenal Actinomycosis in Captive Ball Pythons (Python regius).

Ball pythons (Python regius) are one of the most commonly kept and bred reptiles in captivity. In a large ball python breeding colony, a unique syndrome characterized by granulomatous inflammation of the cloaca and hemipenes (phalli) was observed in 140 of 481 (29.1%) breeding males, but only one of 1,446 breeding females. Lesions were absent in virgin males (n = 201) and virgin females (n = 293). On postmortem examination (n = 13, 12 males, 1 female), numerous well-defined mucosal and submucosal granulomas were present in the hemipenes (males) and cloaca (males and female). Extension into the coelomic cavity and liver was noted in a subset of these animals. An additional small subset of breeder animals (6/2027; 0.3%) presented with oral and mandibular swellings. Postmortem examination (n = 4, all female) showed oral lesions histologically indistinguishable from the cloacal/hemipenal lesions. Aerobic bacterial culture of a hepatic granuloma of one snake resulted in the isolation of filamentous, Gram-positive bacilli; amplification, and sequencing of the 16S rRNA gene and subsequent phylogenetic analysis of the isolate identified the bacterium as a novel species of Actinomyces. Screening of cloacal and oral granulomas using a specific, heminested 16S rRNA PCR assay confirmed the presence of the agent in all 17 snakes, as well as in cloacal swabs taken at the time of necropsy in 11/13 snakes. The Actinomyces sp. was also identified by PCR of cloacal swabs of unaffected snakes (n = 94) from the affected colony and two unrelated, grossly unaffected breeding colonies. In the affected colony, 65.5% of breeding animals (n = 23) but only 11.9% of virgin animals (n = 42) tested PCR positive, with breeding status being a significant predictor of bacterium presence (P < 0.00001). This study characterizes a granulomatous mucosal disease syndrome of breeding male ball pythons associated with a novel Actinomyces. In stark contrast to male snakes, the presence of the bacterium in both breeding and virgin females was very rarely associated with clinical disease. Though additional studies are necessary, these data suggest a role for the novel bacterium in the disease process, a predilection for clinical disease in male snakes, and the potential for sexual transmission of the disease.

Advantages of 16S rRNA PCR for the diagnosis of prosthetic joint infection

16S ribosomal RNA (rRNA) PCR has been reported to be an effective diagnostic means in patients with prosthetic joint infection (PJI). The aim of the present meta-analysis is to establish the overall diagnostic accuracy of the measurement of 16S rRNA PCR for diagnosing PJI. PubMed, Web of Science, Cochrane Library, EMBASE and Wiley Online Library were searched for studies on 16S rRNA PCR in the diagnosis of PJI. The search incorporated all literature published up until December 2018 and the QUADAS-2 checklist were used for quality assessment. The sensitivity, specificity and other measures of accuracy of 16S rRNA PCR in the diagnosis of PJI were pooled. Statistical analysis was performed by employing Meta-Disc 1.4 and Stata 12.0 software. A total of 15 studies met the inclusion criteria. The summary estimates for 16S rRNA PCR in the diagnosis of PJI in these studies were pooled: Sensitivity, 0.70 (95% CI, 0.67-0.73); specificity, 0.93 (95% CI, 0.91-0.94); positive likelihood ratio, 10.93 (95% CI, 5.55-21.51); negative likelihood ratio, 0.33 (95% CI, 0.28-0.40); diagnostic odds ratio, 41.77 (95% CI, 19.90-87.68); and the area under the curve, 0.89. Subgroup analysis showed that the use of sonicate fluid and periprosthetic tissue has higher sensitivity (0.76; 95% CI, 0.69-0.82; and 0.73; 95% CI, 0.68-0.78, respectively), specificity (0.93, 95% CI, 0.90-0.96; and 0.95; 95% CI, 0.90-0.98, respectively) and area under the curve (0.93 and 0.98, respectively). 16S rRNA PCR assay plays an important role in the diagnosis of PJI. The results of 16S rRNA PCR assays should be interpreted in parallel with clinical findings, the results of microbiological, and other laboratory tests.

Detection and characterization of bacterial endosymbionts inSoutheast Asian tephritid fruit fly populations

BackgroundVarious endosymbiotic bacteria, including Wolbachia of the Alphaproteobacteria, infect a wide range ofinsects and are capable of inducing reproductive abnormalities to their hostssuch as cytoplasmic incompatibility (CI), parthenogenesis, feminization andmale-killing. These extended phenotypes can be potentially exploited inenhancing environmentally friendly methods, such as the sterile insect technique(SIT), for controlling natural populations of agricultural pests. The goal ofthe present study is to investigate the presence of Wolbachia, Spiroplasma,Arsenophonus and Cardinium among Bactrocera,Dacus and Zeugodacus flies of Southeast Asian populations, and to genotypeany detected Wolbachia strains.ResultsA specific 16S rRNA PCR assay wasused to investigate the presence of reproductive parasites in naturalpopulations of nine different tephritid species originating from three Asiancountries, Bangladesh, China and India. Wolbachia infections were identified in Bactrocera dorsalis, B.correcta, B. scutellaris andB. zonata, with 12.2–42.9% occurrence,Entomoplasmatales in B. dorsalis, B. correcta, B.scutellaris, B. zonata,Zeugodacus cucurbitae and Z. tau (0.8–14.3%) and Cardinium in B. dorsalis andZ. tau (0.9–5.8%), while none of thespecies tested, harbored infections with Arsenophonus. Infected populations showed a medium (between 10and 90%) or low (< 10%) prevalence, ranging from 3 to 80% for Wolbachia, 2 to 33% for Entomoplasmatales and 5 to45% for Cardinium. Wolbachia and Entomoplasmatales infections were found both intropical and subtropical populations, the former mostly in India and the latterin various regions of India and Bangladesh. Cardinium infections were identified in both countries but onlyin subtropical populations. Phylogenetic analysis revealed the presence ofWolbachia with some strains belongingeither to supergroup B or supergroup A. Sequence analysis revealed deletions ofvariable length and nucleotide variation in three Wolbachia genes. Spiroplasmastrains were characterized as citri–chrysopicola–mirum and ixodetis strainswhile the remaining Entomoplasmatales to the Mycoides–Entomoplasmataceae clade.Cardinium strains were characterized asgroup A, similar to strains infecting Encarsiapergandiella.ConclusionsOur results indicated that in the Southeast natural populationsexamined, supergroup A Wolbachia straininfections were the most common, followed by Entomoplasmatales and Cardinium. In terms of diversity, most strains ofeach bacterial genus detected clustered in a common group. Interestingly, thedeletions detected in three Wolbachia geneswere either new or similar to those of previously identified pseudogenes thatwere integrated in the host genome indicating putative horizontal gene transferevents in B. dorsalis, B. correcta and B.zonata.

2181. Yield and Impact of Molecular Diagnostics for Pathogen Detection in Pediatric Patients: 16/18S rRNA PCR and Noninvasive Assays

BackgroundMolecular diagnostic tests can identify bacterial and fungal pathogens from clinical samples. Nucleic acid detection tests include 16S and 18S rRNA gene PCR (16/18S PCR) and plasma next-generation sequencing (NGS). Other assays (fungal galactomannan and 1,3-β-d-glucan) detect structural factors. Our objective was to assess the utilization, yield, and impact of molecular diagnostics in pediatric patients who had samples sent for 16/18S PCR.MethodsSterile site fluid or tissue specimens were collected as part of standard care at Lurie Children’s Hospital, cultured, and sent to Northwestern Memorial Hospital for 16/18S PCR as clinically indicated. Medical records were reviewed for diagnostics, antibiotics, and clinical course.ResultsFrom 1/2016–8/2018, 236 samples were sent for 16 and/or 18S PCR from 183 patients. 83% had a concurrent ID consult. 16S PCR was done on 215 samples, 42 (20%) were positive, and 36 yielded species identification (Table 1). Antibacterial agents were administered prior to specimen collection in 73% and did not affect likelihood of positive 16S PCR. 18S PCR was sent on 163 samples; 12 (7.4%) were positive (Table 2) of which 10 were from immunocompromised hosts. 40% of patients were on antifungals prior to sample acquisition. 16/18S PCR impacted antimicrobial decision-making in 70 cases (30%). A pathogenic fungus was detected by PCR but not culture in 2 cases. Time to positivity of fungal culture was 1–15 days. Fungal culture was positive in 5 cases with-negative 18S PCR. Seventeen patients had positive serum 1,3-ß-D-glucan and/or galactomannan: 3 of which had positive 18S PCR, 5 with fungal growth, 5 presumed infection based on imaging, 1 Nocardia, and 3 noninfectious etiology. Plasma NGS was sent on 45 cases, was positive in 34, and affected clinical management in 10.Conclusion16S PCR can identify bacterial pathogens in the setting of negative culture and impact clinical care. Abscess, bronchial/pleural fluid, and brain/organ tissue were high yield specimens. 18S PCR can provide expeditious fungal identification in cases of suspected invasive disease, but fungal culture and serum molecular testing increase diagnostic yield. No single fungal test is comprehensive. Plasma NGS had relatively high yield and clinical impact in selected patients. Disclosures All authors: No reported disclosures.

Genetic diversity of Anaplasma and Ehrlichia bacteria found in Dermacentor and Ixodes ticks in Mongolia

Anaplasma and Ehrlichia are tick-borne bacterial pathogens that cause human granulocytic anaplasmosis, human monocytic ehrlichiosis, and are severe threats to livestock economies like Mongolia. In this study, ticks were collected, identified, and pooled (n = 299) from three distinct environments across central Mongolia. Each pool was initially tested for Anaplasma/Ehrlichia using a 16S rRNA PCR assay that detects both genera, and specific PCR testing was done to identify those positive samples. Maximum likelihood estimation (MLE) of infection rates of ticks collected from the environment in Selenge aimag (province) found infection rates of Ixodes persulcatus ticks to be 2.0% (95% CI: 0.7, 4.3%) for A. phagocytophilum and 0.8% (95% CI: 0.1, 2.5%) for both nonspecific Ehrlichia and Anaplasma. Ehrlichia muris was only detected in I. persulcatus ticks collected from the Selenge aimag, where the MLE was 1.2% (95% CI: 0.1, 2.5%). The calculated MLE infection rate of Anaplasma spp. in questing Dermacentor nuttalli ticks ranged from 1.9% (95% CI: 1.1, 9.1%) in the Tov aimag to 2.3% (95% CI: 1.3, 10.8%) in the Selenge aimag. However, when examining MLE in ticks removed from livestock, estimates increase substantially, ranging from 7.8% (95% CI: 4.2, 13.3%) in Dornogovi to 22.5% (95% CI: 14.3, 34.3%) in Selenge, suggesting that livestock play a key role in disease maintenance. Considering the collective economic losses that can result from these pathogens and the potential for illness in nomadic herdsmen, these results highlight the need for enhanced TBD surveillance and prevention measures within Mongolia.

Detection of Borrelia miyamotoi in Ixodes nipponensis in Korea.

BackgroundThis study investigated Borrelia species prevalence in ticks from vegetation, through a molecular method, in Gwangju Metropolitan City, South Korea.Methodology/Principal findingsA total of 484 ticks were collected through flagging and dragging in a suburban area of Gwangju Metropolitan City, South Korea, in 2014. These ticks were morphologically identified and subjected to nested PCR, targeting Borrelia–specific CTP synthase (pyrG), outer surface protein A (ospA) and flagellin (flaB) genes. Molecular biological species identification of Borrelia-positive ticks was conducted via 16S rRNA PCR assays. Of the 484 ticks collected, 417 (86.2%) were identified as Haemaphysalis longicornis, 42 (8.7%) as H. flava, and 25 (5.2%) as Ixodes nipponensis. All the ixodid ticks containing Borrelia species bacteria were confirmed to be I. nipponensis adults, by both morphological and molecular methods. Of the 25 I. nipponensis ticks collected, four (16%) were positive for Borrelia species, three of which were B. afzelii and one B. miyamotoi.Conclusions/SignificanceOur study has shown the harboring of B. miyamotoi by I. nipponensis in South Korea. Morphological and molecular genetic analyses revealed that, in South Korea, I. nipponensis could potentially transmit B. miyamotoi to humans.

Rapid Detection of Pathogenic Bacteria in Whole Blood Samples Using 23S rRNA PCR Assays

Purpose:Bloodstream infections are a general cause of death among hospitalized patients. Rapid diagnosis and timely treatment can reduce mortality. The aim of this investigation was to evaluate the 23S rRNA PCR assays as a rapid detection method for diagnose of sepsis in patients with suspected bacteremia.Methods:A cross-sectional study was conducted at Shahid Beheshti University Hospital in Kashan from November 2017 to December 2018. The blood samples of 265 patients with suspected bacteremia were studied by blood culture and 23S rRNA PCR techniques. The results were analyzed using SPSS version 16 and Chi-square test.Results:Eighty (30.2%) blood samples of 265 suspected patients, were identified as positive by PCR assays, whereas 27 (10.2%) were identified as positive by the blood culture technique. The statistical analysis showed a significant association between the results of PCR assays and blood culture and factors such as prior antibiotic use and underlying diseases (P˂0.05). Also a significant correlation was observed between laboratory and clinical criteria and the results of both PCR assays and blood culture (P˂ 0.05).Conclusion:The 23S rRNA PCR method is a rapid and sensitive technique specially for diagnosing sepsis among patients in whom bacteremia is difficult to diagnose with culture method including neonates and patients who have taken antibiotics before microbial culture.

Chlamydia pecorum gastrointestinal tract infection associations with urogenital tract infections in the koala (Phascolarctos cinereus).

BackgroundChlamydia infects multiple sites within hosts, including the gastrointestinal tract (GIT). In certain hosts, gastrointestinal infection is linked to treatment avoidance and self-infection at disease susceptible sites. GIT C. pecorum has been detected in livestock and koalas, however GIT prevalence rates within the koala are yet to be established.MethodsPaired conjunctival, urogenital and rectal samples from 33 koalas were screened for C. pecorum and C. pecorum plasmid using 16S rRNA and CDS5-specific quantitative PCR assays, respectively. Amplicon sequencing of 359 bp ompA fragment was used to identify site-specific genotypes.ResultsThe overall C. pecorum prevalence collectively (healthy and clinically diseased koalas) was 51.5%, 57.6% and 42.4% in urogenital, conjunctival and gastrointestinal sites, respectively. Concurrent urogenital and rectal Chlamydia was identified in 14 koalas, with no cases of GIT only Chlamydia shedding. The ompA genotype G dominated the GIT positive samples, and genotypes A and E’ were dominant in urogenital tract (UGT) positive samples. Increases in C. pecorum plasmid per C. pecorum load (detected by PCR) showed clustering in the clinically diseased koala group (as assessed by scatter plot analysis). There was also a low correlation between plasmid positivity and C. pecorum infected animals at any site, with a prevalence of 47% UGT, 36% rectum and 40% faecal pellet.ConclusionsGIT C. pecorum PCR positivity suggests that koala GIT C. pecorum infections are common and occur regularly in animals with concurrent genital tract infections. GIT dominant genotypes were identified and do not appear to be related to plasmid positivity. Preliminary results indicated a possible association between C. pecorum plasmid load and clinical UGT disease.

Utility of broad-range 16S rRNA PCR assay versus conventional methods for laboratory diagnosis of bacterial endophthalmitis in a tertiary care hospital

BackgroundEndophthalmitis, a sight-threatening intraocular infection, can be of postsurgical, post-traumatic or endogenous origin. Laboratory diagnosis-based appropriate therapy can be vision-saving. Conventional culture-based laboratory diagnosis takes time and lacks sensitivity. In...

An integrated microfluidic system using mannose-binding lectin for bacteria isolation and biofilm-related gene detection

Molecular diagnosis of biofilm-related genes (BRGs) in common bacteria that cause periprosthetic joint infections may provide crucial information for clinicians. In this study, several BRGs, including ica, fnbA, and fnbB, were rapidly detected (within 1 h) with a new integrated microfluidic system. Mannose-binding lectin (MBL)-coated magnetic beads were used to isolate these bacteria, and on-chip nucleic acid amplification (polymerase chain reaction, PCR) was then performed to detect BRGs. Both eukaryotic and prokaryotic MBLs were able to isolate common bacterial strains, regardless of their antibiotic resistance, and limits of detection were as low as 3 and 9 CFU for methicillin-resistant Staphylococcus aureus and Escherichia coli, respectively, when using a universal 16S rRNA PCR assay for bacterial identification. It is worth noting that the entire process including bacteria isolation by using MBL-coated beads for sample pre-treatment, on-chip PCR, and fluorescent signal detection could be completed on an integrated microfluidic system within 1 h. This is the first time that an integrated microfluidic system capable of detecting BRGs by using MBL as a universal capturing probe was reported. This integrated microfluidic system might therefore prove useful for monitoring profiles of BRGs and give clinicians more clues for their clinical judgments in the near future.

Analysis of the association between density of Helicobacter spp and gastric lesions in dogs.

OBJECTIVE To evaluate the correlation between the density of native gastric Helicobacter spp and the presence of gastric lesions in dogs. ANIMALS 80 dogs of various breeds, sexes, and ages. PROCEDURES Gastroscopic and histologic examinations were performed for all dogs. Helicobacter spp were detected by combining evaluation of urease activity and results of bacteriologic culture, microscopic observation, and a 16S rRNA PCR assay. The density of Helicobacter-like organisms was evaluated with light microscopy by use of Warthin-Starry modified stain. Correlations were evaluated by use of the Spearman correlation analysis. RESULTS Gastritis was found in 55 of 80 dogs and classified as mild (n = 31), moderate (16), or severe (8). Of these 55 dogs, only 8 had clinical signs. Histologic examination revealed some degree of lymphocytic-plasmacytic infiltrate, mild eosinophilia, and neutrophilic inflammation in the lamina propria. Seventy-six dogs had positive results for Helicobacter spp. Helicobacter pylori DNA was not detected. Low density and homogeneous distribution of Helicobacter spp were observed in all gastric zones. CONCLUSIONS AND CLINICAL RELEVANCE A significant correlation between density of Helicobacter spp and gastroscopic or histologic lesions was not detected. These findings supported the contention that there is no correlation between general Helicobacter spp density or numbers and gastritis in dogs.

Persistence of marine fish environmental DNA and the influence of sunlight.

Harnessing information encoded in environmental DNA (eDNA) in marine waters has the potential to revolutionize marine biomonitoring. Whether using organism-specific quantitative PCR assays or metabarcoding in conjunction with amplicon sequencing, scientists have illustrated that realistic organism censuses can be inferred from eDNA. The next step is establishing ways to link information obtained from eDNA analyses to actual organism abundance. This is only possible by understanding the processes that control eDNA concentrations. The present study uses mesocosm experiments to study the persistence of eDNA in marine waters and explore the role of sunlight in modulating eDNA persistence. We seeded solute-permeable dialysis bags with water containing indigenous eDNA and suspended them in a large tank containing seawater. Bags were subjected to two treatments: half the bags were suspended near the water surface where they received high doses of sunlight, and half at depth where they received lower doses of sunlight. Bags were destructively sampled over the course of 87 hours. eDNA was extracted from water samples and used as template for a Scomber japonicus qPCR assay and a marine fish-specific 12S rRNA PCR assay. The latter was subsequently sequenced using a metabarcoding approach. S. japonicus eDNA, as measured by qPCR, exhibited first order decay with a rate constant ~0.01 hr -1 with no difference in decay rate constants between the two experimental treatments. eDNA metabarcoding identified 190 organizational taxonomic units (OTUs) assigned to varying taxonomic ranks. There was no difference in marine fish communities as measured by eDNA metabarcoding between the two experimental treatments, but there was an effect of time. Given the differences in UVA and UVB fluence received by the two experimental treatments, we conclude that sunlight is not the main driver of fish eDNA decay in the experiments. However, there are clearly temporal effects that need to be considered when interpreting information obtained using eDNA approaches.

Assessment of a quantitative 5′ nuclease real-time polymerase chain reaction using groEL gene for Ehrlichia and Anaplasma species in rodents in Brazil

New genotypes of Anaplasmataceae agents have been detected in wild carnivores, birds and deer in Brazil. The present work aimed to investigate the presence of Ehrlichia and Anaplasma species in rodents sampled in Brazil. Additionally, a newly designed quantitative 5′ nuclease real-time multiplex PCR for Ehrlichia and Anaplasma spp. detection based on groEL gene amplification was designed, showing high specificity and sensitivity (10 groEL fragment copy/μL). Between 2000 and 2011, different rodent species [n=60] were trapped in 5 Brazilian biomes. Among 458 rodent spleen samples, 0.4% (2/458) and 2.4% (11/458) were positive for Ehrlichia and Anaplasma spp., respectively. Of 458 samples, 2.0% (9/458) and 1.1% (5/458) were positive for Anaplasma sp. and Ehrlichia sp., respectively, using conventional 16S rRNA PCR assays. Maximum Likelihood phylogenetic analyse based on a small region of 16S rRNA genes positioned the Anaplasma genotypes in rodents near Anaplasma phagocytophilum or Anaplasma marginale and Anaplasma odocoilei isolates. Ehrlichia genotypes were closely related to E. canis. There was a low occurrence of Anaplasma and Ehrlichia in wild and synanthropic rodents in Brazil, suggesting the circulation of new genotypes of these agents in rodents in the studied areas.