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Volume 2,
Issue 3,
2020
Volume 2, Issue 3, 2020
- Research Article
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Temperature-gradient incubation isolates multiple competitive species from a single environmental sample
More LessHigh-throughput sequencing has allowed culture-independent investigation into a wide variety of microbiomes, but sequencing studies still require axenic culture experiments to determine ecological roles, confirm functional predictions and identify useful compounds and pathways. We have developed a new method for culturing and isolating multiple microbial species with overlapping ecological niches from a single environmental sample, using temperature-gradient incubation. This method was more effective than standard serial dilution-to-extinction at isolating methanotrophic bacteria. It also highlighted discrepancies between culture-dependent and -independent techniques; 16S rRNA gene amplicon sequencing of the same sample did not accurately reflect cultivatable strains using this method. We propose that temperature-gradient incubation could be used to separate out and study previously ‘unculturable’ strains, which co-exist in both natural and artificial environments.
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Expression changes of cytotoxicity and apoptosis genes in HTLV-1-associated myelopathy/tropical spastic paraparesis patients from the perspective of system virology
More LessAlthough human T-cell lymphotropic virus type-1 (HTLV-1) was the first retrovirus among human pathogens to be identified, insufficient information on the pathogenesis of HTLV-1 infection means that no precise mechanism has yet been provided for HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Based on previous studies, it was found that apoptosis and inflammation stimulation were among the most important mechanisms underlying HAM/TSP. The present study provides an in-silico analysis of the microarray data related to HAM/TSP patients. Expression changes of the genes responsible for cytotoxicity and apoptosis processes of HAM/TSP patients and asymptomatic carriers were investigated. Expression of the genes involved in cytotoxicity and apoptosis in HAM/TSP patients was decreased; hence, a model was proposed indicating that the spread of immune responses in HAM/TSP may be due to expression of HTLV-1 virulence factors and the resistance of HTLV-1-infected cells to apoptosis.
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Diagnosis of Acute Gastroenteritis with Immunochromatography and Effectiveness of Rotavirus Vaccine in a Japanese Clinic
Despite the well known effectiveness of two licensed live attenuated oral rotavirus (RV)-vaccines, Rotarix and RotaTeq, constant monitoring of vaccine effectiveness (VE) is essential considering the evolving power and reassortment capability of RVs. In this study, we detected RV, norovirus (NV) and adenovirus (AV) infections using immunochromatography (IC)-based kits in children with acute gastroenteritis (AGE) who attended a pediatric clinic in Kiryu city, Gunma, Japan during June, 2014–September, 2018. VEs were determined using a test-negative study design. Among 1658 AGE-children, RV, NV and AV were detected in 96 (5.8 %), 146 (8.8 %) and 46 (2.8 %) children, respectively. Interestingly, the distributions of infections were found to be associated with age and sex. Namely, RV infections were significantly higher in female (P=0.02) and in the 19–30 month age group children, while NV and AV infections predominated in the 13–24 month and 7–18 month age groups, respectively. The disease severity for RV and NV infections remained similar and significantly higher than that of AV infections. The VE of RV-vaccines was 49.8 % (95 % CI: 22.7 to 67.3 %) against all RV infections, which was increased up to 67.2 % (95 % CI: 35.3 to 83.4 %) against severe RV infections. RV-vaccinated children experienced less severe symptoms in RV-infections while non-RV AGE remained less serious for both RV-vaccinated and unvaccinated children. Finally, the prevalence of RV infection remained minimized (≤5.4 %) in this population since 2015. Thus, this study provided important information on distribution of major AGEs in young children and exhibited the effective role of RV vaccines in post-vaccine era.
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Principal component analysis exploring the association between antibiotic resistance and heavy metal tolerance of plasmid-bearing sewage wastewater bacteria of clinical relevance
More LessThis paper unravels the occurrence of plasmid-mediated antibiotic resistance in association with tolerance to heavy metals among clinically relevant bacteria isolated from sewage wastewater. The bacteria isolated were identified following conventional phenotypic and/or molecular methods, and were subjected to multiple-antibiotic resistance (MAR) profiling. The isolates were tested against the heavy metals Hg2+, Cd2+, Cr2+ and Cu2+. SDS-PAGE and agarose gel electrophoretic analyses were performed, respectively, for the characterization of heavy metal stress protein and R-plasmid among the isolated bacteria. Principal component analysis was applied in determining bacterial resistance to antibiotics and heavy metals. Both lactose-fermenting ( Escherichia coli ) and non-fermenting ( Acinetobacter baumannii and Pseudomonas putida ) Gram-negative bacterial strains were procured, and showed MAR phenotypes with respect to three or more antibiotics, along with resistance to the heavy metals Hg2+, Cd2+, Cr2+ and Cu2+. The Gram-positive bacteria, Enterococcus faecalis , isolated had ‘ampicillin–kanamycin–nalidixic acid’ resistance. The bacterial isolates had MAR indices of 0.3–0.9, indicating their ( E. faecalis , E. coli , A. baumannii and P. putida ) origin from niches with high antibiotic pollution and human faecal contamination. The Gram-negative bacteria isolated contained a single plasmid (≈54 kb) conferring multiple antibiotic resistance, which was linked to heavy metal tolerance; the SDS-PAGE analysis demonstrated the expression of heavy metal stress proteins (≈59 and ≈10 kDa) in wastewater bacteria with a Cd2+ stressor. The study results grant an insight into the co-occurrence of antibiotic resistance and heavy metal tolerance among clinically relevant bacteria in sewage wastewater, prompting an intense health impact over antibiotic usage.
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- Short Communication
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Comparing conventional, biochemical and genotypic methods for accurate identification of Klebsiella pneumoniae in Sudan
More LessKlebsiella pneumoniae is recognized as one of the most important healthcare-associated pathogens worldwide due to its tendency to develop antibiotic resistance and cause fatal outcomes. Bacterial identification methods such as culture and biochemical tests are routinely used with limited accuracy in many low- and middle-income countries, including Sudan. The aim of this study was to test the accuracy of identification of K. pneumoniae in Khartoum, Sudan. Two hundred and fifty K. pneumoniae isolates were collected and identified using conventional phenotypic methods, biochemically using API 20E and genotypically by amplification of 16S−23S rDNA and sequencing of rpoB, gapA and pgi. Only 139 (55.6 %) of the isolates were confirmed as K. pneumoniae genotypically by PCR and 44.4 % were identified as non- K. pneumoniae . The results demonstrate that the identification panels used by the hospitals were inaccurately identifying K. pneumonia and led to overestimation of the prevalence of this organism. The current identification methods used in Khartoum hospitals are highly inaccurate, and therefore we recommend the use of a comprehensive biochemical panel or molecular methods, when possible, for accurate identification of K. pneumoniae .
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What is the role of the thioredoxin antioxidant complex in relation to HAM/TSP?
More LessWe have little information about the definite role of the thioredoxin antioxidant complex system during viral infection, particularly during human T-cell lymphotropic virus type 1 (HTLV-1) infection and the HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) state. Therefore, we conducted comprehensive next-generation sequencing (NGS) analysis to determine Trx system expression changes in three categories of subjects: sero-negative HTLV-1 individuals, asymptomatic HTLV-1 people and HAM/TSP patients. We found that Trx capacity is reduced in the HAM/TSP state compared to healthy individuals, which indicates increasing inflammation and reduction of apoptosis, which might contribute to the progression of inflammation in the spinal cord, which in turn may develop into the HAM/TSP state.
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Laboratory methods supporting measles surveillance in Queensland, Australia, 2010–2017
Purpose. Australia was officially recognised as having eliminated endemic measles transmission in 2014. Maintaining laboratory support for surveillance of vaccine-preventable diseases, such as measles, is an essential component of reaching and maintaining transmission-free status.
Methodology. Real-time and conventional PCR-based tools were used to detect, differentiate from measles vaccine virus (MeVV), and sequence fragments of measles viruses (MeV) identified from specimens collected in Queensland. Specimens were mostly from travellers who had visited or returned to Queensland from international or interstate sites or been in contact with a case from either group.
Results. Between 2010 and 2017, 13 678 specimens were tested in our laboratory using real-time RT-PCR (RT-rPCR), identifying 533 positives. Most specimens were swabs (70.98 %) and urines (25.56 %). A MeVV RT-rPCR was used on request and identified 154 instances of MeVV. MeV-positive extracts were genotyped as required. Genotypes identified among sequenced specimens included B3, D4, D8, D9, G3, and H1 as well as members of clade A as expected from the detection of MeV among virus introductions due to global travel and vaccination.
Conclusion. We describe the workflow employed and results from our laboratory between 2010 and 2017 for the sensitive detection of MeV infection, supporting high-quality surveillance to ensure the maintenance of Australia’s measles-free status.
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- Method
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Re-validation and update of an extended-specificity multiplex assay for detection of Streptococcus pneumoniae capsular serotype/serogroup-specific antigen and cell-wall polysaccharide in urine specimens
National surveillance of pneumococcal disease at the serotype level is essential to assess the effectiveness of vaccination programmes. We previously developed a highly sensitive extended-specificity multiplex immunoassay for detection of Streptococcus pneumoniae serotype-specific antigen in urine in the absence of isolates. The assay uses human mAbs that detect the 24 pneumococcal serotype/groups targeted by the pneumococcal conjugate vaccines (PCVs) and pneumococcal polysaccharide vaccine (PPV-23) plus some cross-reactive types and the pneumococcal cell-wall polysaccharide. However, the previous assay had some limitations, namely the reduced specificity of the serotype 7F, 20 and 22F assays, for which non-specific binding in urine samples was observed. Here we report on the further development and re-validation of a new version of the assay (version 2.1), which offers improved sensitivity towards serotypes 7F, 18C and 19F and increased specificity for serotypes 7F, 20 and 22F by replacement of some of the antibody clones with new clones. Using a panel of urine specimens from patients diagnosed with community-acquired pneumonia or pneumococcal disease, the overall clinical sensitivity of this version of the assay based on isolation of S. pneumoniae from a normally sterile site is 94.3 % and the clinical specificity is 93.6 %, in comparison with clinical sensitivity and specificity values of 96.2 % and 89.9 % in the previous assay.
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- Case Report
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A descriptive case of persistent Acanthamoeba keratitis: raising awareness of this complex ocular disease
More LessBackground. Acanthamoeba species are ubiquitous free-living organisms found in the environment. They can cause a sight-threatening disease of the cornea termed Acanthamoeba keratitis (AK), often associated with contact-lens wearers. This case review describes a persistent presentation of AK and raises awareness of the challenges faced when diagnosing and managing the disease. It highlights the importance of an accurate and rapid diagnosis to assist patient management and to maximize the potential for a better outcome.
Case presentation. A 73-year-old female was admitted to hospital due to vision impairment of her left eye. Following a clinical examination, the diagnosis of herpes simplex keratitis (HSK) was reported and treated with antivirals. However, deterioration of her keratitis continued after initial treatment, which prompted an investigation into the possibility of AK. Molecular testing of sequential corneal tissue was performed using a real-time PCR assay alongside further clinical examinations. Acanthamoeba species DNA was isolated from seven out of eight corneal tissues over a 12 month period. Following prolonged drug treatment and two corneal transplants, the individual’s symptoms ceased and further molecular testing of corneal tissue was negative.
Conclusions. Acanthamoeba keratitis can be easily misdiagnosed due to the similarities in the clinical presentation to other, much more common ocular pathogens. This case highlights the importance of considering AK in the first-line diagnosis, and raises awareness that an early, accurate and rapid diagnosis is crucial to improve patient outcome.
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The first case of HIV-2 in Scotland
More LessHIV-1 infects an estimated 37 million people worldwide, while the rarer HIV-2 infects 1–2 million worldwide. HIV-2 is mainly restricted to West African countries. The majority of patients in Scotland are diagnosed with HIV-1, but in 2013 the West of Scotland Specialist Virology Centre (WoSSVC) diagnosed Scotland’s first HIV-2 positive case in a patient from Côte d’Ivoire. HIV-2 differs from HIV-1 in terms of structural viral proteins, viral transmissibility, prolonged period of latency, intrinsic resistance to certain antivirals and how to monitor the effectiveness of treatment. Over the course of 5 years the patient has required several changes in treatment due to both side effects and pill burden. This case highlights the complexity of HIV-2 patient management over time.
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