RNomic identification and evaluation of npcTB_6715, a non-protein-coding RNA gene as a potential biomarker for the detection of Mycobacterium tuberculosis.

Technological advances in RNA biology greatly improved transcriptome profiling during the last two decades. Besides the discovery of many small RNAs (sRNA) that are involved in the physiological and pathophysiological regulation of various cellular circuits, it becomes evident that the corresponding RNA genes might also serve as potential biomarkers to monitor the progression of disease and treatment. sRNA gene candidate npcTB_6715 was previously identified via experimental RNomic (unpublished data), and we report its application as potential biomarker for the detection of Mycobacterium tuberculosis (MTB) in patient samples. For proof of principle, we developed a multiplex PCR assay and report its validation with 500 clinical cultures, positive for Mycobacteria. The analysis revealed 98.9% sensitivity, 96.1% specificity, positive and negative predictive values of 98.6% and 96.8%, respectively. These results underscore the diagnostic value of the sRNA gene as diagnostic marker for the specific detection of MTB in clinical samples. Its successful application and the general ease of PCR-based detection compared to standard bacterial culture techniques might be the first step towards 'point-of-care' diagnostics of Mycobacteria. To the best of our knowledge, this is the first time for the design of diagnostic applications based on sRNA genes, in Mycobacteria.

Risk factors and clinical outcome of profound thrombocytopenia in adult patients with DENV infections.

Severe thrombocytopenia is common in dengue virus (DENV) infections. However, studies focusing on the role of profound thrombocytopenia (PT) (nadir platelet counts ≤ 20 000/mm3) in DENV infections are scarce. This study aims to identify the associated features and outcome of DENV patients with PT. It involves 237 adult hospitalized patients who were DENV PCR positive. The presence of comorbidity (AOR = 4.625; 95% CI = 1.113-19.230), higher admission hematocrit (AOR = 1.213; 95% CI = 1.067-1.379), lower admission albumin (AOR = 0.870; 95% CI = 0.766-0.988) and lower admission platelets (AOR = 0.980; 95% CI = 0.969-0.991) was associated with platelets ≤ 20 000/mm3 in multivariate logistic regression. PT was not affected by DENV serotypes, coinfections and secondary DENV infections. Patients with PT had significantly higher risk of experiencing warning signs (AOR = 3.709, 95% CI = 1.089-12.634) and longer hospital stay (AOR = 1.943, 95% CI = 1.010-3.774). However, severe dengue disease, hemorrhagic manifestations and need for intensive care were not significantly associated with PT.

Impact of dengue virus (DENV) co-infection on clinical manifestations, disease severity and laboratory parameters.

BACKGROUND: The co-circulation of 4 DENV serotypes in geographically expanding area, has resulted in increasing occurrence of DENV co-infections. However, studies assessing the clinical impact of DENV co-infections have been scarce and have involved small number of patients. This study explores the impact of DENV co-infection on clinical manifestations and laboratory parameters. METHODS: This retrospective study involved consecutive hospitalized patients with non-structural protein 1 (NS1) antigen positivity during an outbreak (Jan to April 2014). Multiplex RT-PCR was performed directly on NS1 positive serum samples to detect and determine the DENV serotypes. All PCR-positive serum samples were inoculated onto C6/36 cells. Multiplex PCR was repeated on the supernatant of the first blind passage of the serum-infected cells. Random samples of supernatant from the first passage of C6/36 infected cells were subjected to whole genome sequencing. Clinical and laboratory variables were compared between patients with and without DENV co-infections. RESULTS: Of the 290 NS1 positive serum samples, 280 were PCR positive for DENV. Medical notes of 262 patients were available for analysis. All 4 DENV serotypes were identified. Of the 262 patients, forty patients (15.3 %) had DENV co-infections: DENV-1/DENV-2(85 %), DENV-1/DENV-3 (12.5 %) and DENV-2/DENV-3 (2.5 %). Another 222 patients (84.7 %) were infected with single DENV serotype (mono-infection), with DENV- 1 (76.6 %) and DENV- 2 (19.8 %) predominating. Secondary dengue infections occurred in 31.3 % patients. Whole genome sequences of random samples representing DENV-1 and DENV-2 showed heterogeneity amongst the DENVs. Multivariate analysis revealed that pleural effusion and the presence of warning signs were significantly higher in the co-infected group, both in the overall and subgroup analysis. Diarrhoea was negatively associated with co-infection. Additionally, DENV-2 co-infected patients had higher frequency of patients with severe thrombocytopenia (platelet count < 50,000/mm(3)), whereas DENV-2 mono-infections presented more commonly with myalgia. Elevated creatinine levels were more frequent amongst the co-infected patients in univariate analysis. Haemoconcentration and haemorrhagic manifestations were not higher amongst the co-infected patients. Serotypes associated with severe dengue were: DENV-1 (n = 9), DENV-2 (n = 1), DENV-3 (n = 1) in mono-infected patients and DENV-1/DENV-2 (n = 5) and DENV-1/DENV-3 (n = 1) amongst the co-infected patients. CONCLUSION: DENV co-infections are not uncommon in a hyperendemic region and co-infected patients are skewed towards more severe clinical manifestations compared to mono-infected patients.

Epidemiology and clinical characteristics of hospitalized patients with pandemic influenza A (H1N1) 2009 infections: the effects of bacterial coinfection.

BACKGROUND: Numerous reports have described the epidemiological and clinical characteristics of influenza A (H1N1) 2009 infected patients. However, data on the effects of bacterial coinfection on these patients are very scarce. Therefore, this study explores the impact of bacterial coinfection on the clinical and laboratory parameters amongst H1N1 hospitalized patients. FINDINGS: This retrospective study involved hospitalized patients with laboratory-confirmed H1N1 infections (September 2009 to May 2010). Relevant clinical data and the detection of bacterial coinfection from respiratory or sterile site samples were obtained. Multiplex PCR was used to determine the co-existence of other respiratory viruses. Comparison was made between patients with and without bacterial coinfection. The occurrence of coinfection was 34%; 14 (28%) bacterial and only 3 (6%) viral. Mycoplasma pneumoniae (n = 5) was the commonest bacteria followed by Staphylococcus aureus (n = 3). In univariate analysis, clinical factors associated with bacterial coinfection were age > 50 years (p = 0.02), presence of comorbidity (p = 0.04), liver impairment (p = 0.02), development of complications (p = 0.004) and supplemental oxygen requirement (p = 0.02). Leukocytosis (p = 0.02) and neutrophilia (p = 0.004) were higher in bacterial coinfected patients. Multivariate logistic regression analysis revealed that age > 50 years and combined complications were predictive of bacterial coinfection. CONCLUSIONS: Bacterial coinfection is not uncommon in H1N1 infected patients and is more frequently noted in the older aged patients and is associated with higher rates of complications. Also, as adjunct to clinical findings, clinicians need to have a higher index of suspicion if neutrophilia was identified at admission as it may denote bacterial coinfection.

Diagnostic accuracy of fresh drooled saliva for SARS-CoV-2 in travelers.

BACKGROUND: The standard for SARS-CoV-2 diagnosis is RT-PCR from nasopharyngeal or oropharyngeal swabs. Major airports require COVID-19 screening, and saliva has the potential as a substitute specimen for SARS-CoV-2 diagnosis. We investigated the utility of fresh drooled saliva against NPS for COVID-19 screening of travelers. METHODS: We recruited 81 travelers and 15 non-travelers (including ten controls) prospectively within a mean of 3·22 days of RT-PCR confirmed COVID-19. Each study participant provided 2 mls of early morning fresh drooled whole saliva separately into a sterile plastic container and GeneFiX™ saliva collection kit. The saliva specimens were processed within 4 h and tested for SARS-CoV-2 genes (E, RdRP, and N2) and the results compared to paired NPS RT-PCR for diagnostic accuracy. RESULTS: Majority of travellers were asymptomatic (75·0%) with a mean age of 34·26 years. 77 travelers were RT-PCR positive at the time of hospitalization whilst three travelers had positive contacts. In this group, the detection rate for SARS-CoV-2 with NPS, whole saliva, and GeneFiX™ were comparable (89·3%, 50/56; 87·8%, 43/49; 89·6%, 43/48). Both saliva collection methods were in good agreement (Kappa = 0·69). There was no statistical difference between the detection rates of saliva and NPS (p > 0·05). Detection was highest for the N2 gene whilst the E gene provided the highest viral load (mean = 27·96 to 30·10, SD = 3·14 to 3·85). Saliva specimens have high sensitivity (80·4%) and specificity (90·0%) with a high positive predictive value of 91·8% for SARS-CoV-2 diagnosis. CONCLUSION: Saliva for SARS-CoV-2 screening is a simple accurate technique comparable with NPS RT-PCR.

Whole-Genome Sequences of Two Carbapenem-Resistant Klebsiella quasipneumoniae Strains Isolated from a Tertiary Hospital in Johor, Malaysia.

We report the whole-genome sequences of two carbapenem-resistant clinical isolates of Klebsiella quasipneumoniae subsp. similipneumoniae obtained from two different patients. Both strains contained three different extended-spectrum ß-lactamase genes and showed strikingly high pairwise average nucleotide identity of 99.99% despite being isolated 3 years apart from the same hospital.

The Complete Sequence and Comparative Analysis of a Multidrug-Resistance and Virulence Multireplicon IncFII Plasmid pEC302/04 from an Extraintestinal Pathogenic Escherichia coli EC302/04 Indicate Extensive Diversity of IncFII Plasmids.

Extraintestinal pathogenic Escherichia coli (ExPEC) that causes extraintestinal infections often harbor plasmids encoding fitness traits such as resistance and virulence determinants that are of clinical importance. We determined the complete nucleotide sequence of plasmid pEC302/04 from a multidrug-resistant E. coli EC302/04 which was isolated from the tracheal aspirate of a patient in Malaysia. In addition, we also performed comparative sequence analyses of 18 related IncFIIA plasmids to determine the phylogenetic relationship and diversity of these plasmids. The 140,232 bp pEC302/04 is a multireplicon plasmid that bears three replication systems (FII, FIA, and FIB) with subtype of F2:A1:B1. The plasmid is self-transmissible with a complete transfer region. pEC302/04 also carries antibiotic resistance genes such as bla TEM-1 and a class I integron containing sul1, cml and aadA resistance genes, conferring multidrug resistance (MDR) to its host, E. coli EC302/04. Besides, two iron acquisition systems (SitABCD and IutA-IucABCD) which are the conserved virulence determinants of ExPEC-colicin V or B and M (ColV/ColBM)-producing plasmids were identified in pEC302/04. Multiple toxin-antitoxin (TA)-based addiction systems (i.e., PemI/PemK, VagC/VagD, CcdA/CcdB, and Hok/Sok) and a plasmid partitioning system, ParAB, and PsiAB, which are important for plasmid maintenance were also found. Comparative plasmid analysis revealed only one conserved gene, the repA1 as the core genome, showing that there is an extensive diversity among the IncFIIA plasmids. The phylogenetic relationship of 18 IncF plasmids based on the core regions revealed that ColV/ColBM-plasmids and non-ColV/ColBM plasmids were separated into two distinct groups. These plasmids, which carry highly diverse genetic contents, are also mosaic in nature. The atypical combination of genetic materials, i.e., the MDR- and ColV/ColBM-plasmid-virulence encoding regions in a single ExPEC plasmid is rare but of clinical importance. Such phenomenon is bothersome when the plasmids are transmissible, facilitating the spread of virulence and resistance plasmids among pathogenic bacteria. Notably, certain TA systems are more commonly found in particular ExPEC plasmid types, indicating the possible relationships between certain TA systems and ExPEC pathogenesis.

Endemicity of Acinetobacter calcoaceticus-baumannii Complex in an Intensive Care Unit in Malaysia.

Introduction. Acinetobacter calcoaceticus-baumannii complex (ACB complex) is a leading opportunistic pathogen in intensive care units (ICUs). Effective control of spread requires understanding of its epidemiological relatedness. This study aims to determine the genetic relatedness and antibiotic susceptibilities of ACB complex in an ICU in Malaysia. Methodology. Pulsed field gel electrophoresis (PFGE), E-test, and disk diffusion were used for isolates characterization. Results. During the study period (December 2011 to June 2012), 1023 patients were admitted to the ICU and 44 ACB complex (blood, n = 21, and blind bronchial aspirates, n = 23) were recovered from 38 ICU patients. Six isolates were from non-ICU patients. Of the 44 ICU isolates, 88.6% exhibited multidrug-resistant (MDR) patterns. There was high degree of resistance, with minimum inhibitory concentration90 (MIC90) of >32 µg/mL for carbapenems and ≥256 µg/mL for amikacin, ampicillin/sulbactam, and cefoperazone/sulbactam. Isolates from the main PFGE cluster were highly resistant. There was evidence of dissemination in non-ICU wards. Conclusion. High number of clonally related MDR ACB complex was found. While the ICU is a likely reservoir facilitating transmission, importation from other wards may be important contributor. Early identification of strain relatedness and implementation of infection control measures are necessary to prevent further spread.