Engineered Mycobacterium tuberculosis antigen assembly into core-shell nanobeads for diagnosis of tuberculosis.

Despite recent advances in diagnosis, tuberculosis (TB) remains one of the ten leading causes of death worldwide. Here, we engineered Mycobacterium tuberculosis (Mtb) proteins (ESAT6, CFP10, and MTB7.7) to self-assemble into core-shell nanobeads for enhanced TB diagnosis. Respective purified Mtb antigen-coated polyester beads were characterized and their functionality in TB diagnosis was tested in whole blood cytokine release assays. Sensitivity and specificity were studied in 11 pulmonary TB patients (PTB) and 26 healthy individuals composed of 14 Tuberculin Skin Test negative (TSTn) and 12 TST positive (TSTp). The production of 6 cytokines was determined (IFNγ, IP10, IL2, TNFα, CCL3, and CCL11). To differentiate PTB from healthy individuals (TSTp + TSTn), the best individual cytokines were IL2 and CCL11 (>80% sensitivity and specificity) and the best combination was IP10 + IL2 (>90% sensitivity and specificity). We describe an innovative approach using full-length antigens attached to biopolyester nanobeads enabling sensitive and specific detection of human TB.

Portable electrochemical immunosensor for detection of Mycobacterium tuberculosis secreted protein CFP10-ESAT6 in clinical sputum samples.

An early detection of Mycobacterium tuberculosis is very important to reduce the number of fatal cases and allow for fast recovery. However, the interpretation of the result from smear microscopy requires skilled personnel due to the propensity of the method to produce false-negative results. In this work, a portable, rapid, and simple sandwich-type immunosensor reader has been developed that is able to detect the presence of M. tuberculosis in sputum samples. By using sandwich-type immunosensor, an anti-CFP10-ESAT6 antibody was immobilized onto the graphene/polyaniline (GP/PANI)-modified gold screen-printed electrode. After incubation with the target CFP10-ESAT6 antigen, the iron/gold magnetic nanoparticles (Fe3O4/Au MNPs) conjugated with anti-CFP10-ESAT6 antibody were used to complete the sandwich format. Differential pulse voltammetry (DPV) technique was used to detect the CFP10-ESAT6 antigen at the potential range of 0.0-1.0 V. The detection time is less than 2 h. Under optimal condition, CFP10-ESAT6 antigen was detected in a linear range from 10 to 500 ng mL-1 with a limit of detection at 1.5 ng mL-1. The method developed from this process was then integrated into a portable reader. The performance of the sensor was investigated and compared with the standard methods (culture and smear microscopy). It provides a good correlation (100% sensitivity and 91.7% specificity) with both methods of detection for M. tuberculosis in sputum samples henceforth, demonstrating the potential of the device as a more practical screening tool.Graphical abstract.

Sandwich Electrochemical Immunosensor for Early Detection of Tuberculosis Based on Graphene/Polyaniline-Modified Screen-Printed Gold Electrode.

A rapid and sensitive sandwich electrochemical immunosensor was developed based on the fabrication of the graphene/polyaniline (GP/PANI) nanocomposite onto screen-printed gold electrode (SPGE) for detection of tuberculosis biomarker 10-kDa culture filtrate protein (CFP10). The prepared GP/PANI nanocomposite was characterized using Fourier transform infrared spectroscopy (FTIR) and field emission scanning electron microscopy (FESEM). The chemical bonding and morphology of GP/PANI-modified SPGE were studied by Raman spectroscopy and FESEM coupled with energy dispersive X-ray spectroscopy, respectively. From both studies, it clearly showed that GP/PANI was successfully coated onto SPGE through drop cast technique. Cyclic voltammetry was used to study the electrochemical properties of the modified electrode. The effective surface area for GP/PANI-modified SPGE was enhanced about five times compared with bare SPGE. Differential pulse voltammetry was used to detect the CFP10 antigen. The GP/PANI-modified SPGE that was fortified with sandwich type immunosensor exhibited a wide linear range (20⁻100 ng/mL) with a low detection limit of 15 ng/mL. This proposed electrochemical immunosensor is sensitive, low sample volume, rapid and disposable, which is suitable for tuberculosis detection in real samples.

The dangers of accepting a single diagnosis: case report of concurrent Plasmodium knowlesi malaria and dengue infection.

BACKGROUND: Dengue and malaria are two common, mosquito-borne infections, which may lead to mortality if not managed properly. Concurrent infections of dengue and malaria are rare due to the different habitats of its vectors and activities of different carrier mosquitoes. The first case reported was in 2005. Since then, several concurrent infections have been reported between the dengue virus (DENV) and the malaria protozoans, Plasmodium falciparum and Plasmodium vivax. Symptoms of each infection may be masked by a simultaneous second infection, resulting in late treatment and severe complications. Plasmodium knowlesi is also a common cause of malaria in Malaysia with one of the highest rates of mortality. This report is one of the earliest in literature of concomitant infection between DENV and P. knowlesi in which a delay in diagnosis had placed a patient in a life-threatening situation. CASE PRESENTATION: A 59-year old man staying near the Belum-Temengor rainforest at the Malaysia-Thailand border was admitted with fever for 6 days, with respiratory distress. His non-structural protein 1 antigen and Anti-DENV Immunoglobulin M tests were positive. He was treated for severe dengue with compensated shock. Treating the dengue had so distracted the clinicians that a blood film for the malaria parasite was not done. Despite aggressive supportive treatment in the intensive care unit (ICU), the patient had unresolved acidosis as well as multi-organ failure involving respiratory, renal, liver, and haematological systems. It was due to the presentation of shivering in the ICU, that a blood film was done on the second day that revealed the presence of P. knowlesi with a parasite count of 520,000/µL. The patient was subsequently treated with artesunate-doxycycline and made a good recovery after nine days in ICU. CONCLUSIONS: This case contributes to the body of literature on co-infection between DENV and P. knowlesi and highlights the clinical consequences, which can be severe. Awareness should be raised among health-care workers on the possibility of dengue-malaria co-infection in this region. Further research is required to determine the real incidence and risk of co-infection in order to improve the management of acute febrile illness.

Comparative study of IgA VH 3 gene usage in healthy TST(-) and TST(+) population exposed to tuberculosis: deep sequencing analysis.

The acquired immune response against tuberculosis is commonly associated with T-cell responses with little known about the role of B cells or antibodies. There have been suggestions that B cells and humoral immunity can modulate the immune response to Mycobacterium tuberculosis. However, the mechanisms involving B-cell responses in M. tuberculosis are not fully understood, in particular the antibody gene preferences. We hypothesized that a preferential use of V genes can be seen associated with resistance to infection mainly in the IgA isotype, which is of prominent importance for infection by pathogens via the mucosal route. We studied healthy individuals with long-term exposure to tuberculosis, infected (TST(+) ) and uninfected TST(-) ) with M. tuberculosis. From a total of 22 V genes analysed, the TST(-) population preferred the VH 3-23 and Vκ1 genes. The VH 3-23 genes were subsequently subjected to 454 amplicon sequencing. The TST(-) population showed a higher frequency of the D3-10 segment compared with the D3-22 segment for the TST(+) population. The J segment usage pattern was similar for both populations with J4 segment being used the most. A preferential pairing of J4 segments to D3-3 was seen for the TST(-) population. The antibodyome difference between both populations suggests a preference for antibodies with VH 3-23, D3-3, JH 4 gene usage by the TST(-) population that could be associated with resistance to infection with M. tuberculosis.

Evaluation of specific humoral immune response and cross reactivity against Mycobacterium tuberculosis antigens induced in mice immunized with liposomes composed of total lipids extracted from Mycobacterium smegmatis.

The development of a new tuberculosis (TB) vaccine has become one of the main objectives of the scientific community. Protein antigens have been widely explored as subunit TB vaccines, however lipid antigens could be equally important to be used or included in such a vaccine. The aim of this study was to demonstrate the potential of a liposome formulation composed of an extract of lipids from Mycobacterium smegmatis (Ms) as a TB vaccine candidate. We evaluated the immunogenicity of this formulation as well as the cross reactive response against antigens from Mycobacterium tuberculosis (MTb) in BALB/c mice. We determined the anti-liposome IgG response in sera from TB patients and from healthy subjects who displayed a positive (PPD+) or negative (PPD-) tuberculin skin test. A significant increase in anti-liposome IgG (p<0.05) was detected in animals immunized with Bacille Calmette-Guérin (BCG) compared with all groups, and in the group immunized with liposomes from Ms (LMs) compared to animals immunized with either LMs adjuvanted with aluminium (LMs-A) or the negative control group (phosphate buffered saline, PBS) respectively. With respect to the cross reactive response against a cocktail of cell wall antigens (CWA) from MTb, significantly higher IgG levels were observed in animals immunized with BCG and LMs compared to negative controls and either, aluminium-adjuvanted liposomes (LMs-A) or montanide (LMs-M) (p<0.05). Furthermore, the anti-liposome IgG response was significantly superior in sera from pulmonary TB patients compared to PPD+ and PPD- healthy subjects (p<0.001) suggesting the expression of these antigens in vivo during active MTb infection. The results obtained provide some evidence for the potential use of liposomes containing total lipid extracts of Ms as a TB vaccine candidate.

Diagnostic performance of an in-house multiplex PCR assay and the retrospective surveillance of bacterial respiratory pathogens at a teaching hospital, Kelantan, Malaysia.

Respiratory tract infections (RTIs), including pneumonia and pulmonary tuberculosis, are among the leading causes of death worldwide. The use of accurate diagnostic tests is crucial to initiate proper treatment and therapy to reduce the mortality rates for RTIs. A PCR assay for simultaneous detection of six respiratory bacteria: Haemophilus influenzae, Klebsiella pneumoniae, Mycobacterium tuberculosis, Pseudomonas aeruginosa, Staphylococcus aureus and Streptococcus pneumoniae, was developed in our lab. The current study aimed to evaluate the performance of this assay along with the retrospective surveillance of respiratory pathogens at a teaching hospital in Kelantan, Malaysia. Leftover sputa (n = 200) from clinical laboratories were collected and undergone DNA template preparation for PCR analysis. Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of the PCR assay were determined in comparison with the gold standard sputum culture. Overall, the accuracy performance of this assay was 94.67% (95% CI: 90.87% to 97.21%) with sensitivity, specificity, PPV and NPV of 100%, 91.67%, 87.1% and 100%, respectively. Based on the organisms detected from sputa, K. pneumoniae ranked as the top isolate (n = 48), followed by P. aeruginosa (n = 13) and H. influenzae (n = 10). Surveillance among the patients showed that the associations of bacterial positive with gender and means of acquisition were found significant (p values = 0.049 and 0.001, respectively). Besides the promising performance of this ready-to-use molecular-based assay for the rapid detection of selected bacteria pathogens, this study also highlighted significant spread of K. pneumoniae RTIs in the community.

Diagnostic detection of intended bacteria associated with respiratory tract infections among Kelantanese Malaysian Hajj pilgrims by a ready-to-use, thermostable multiplex PCR assay.

Bacterial respiratory tract infections (RTIs) are prone to be associated with serious health problems during the annual Hajj pilgrimage and are a public health concern due to the potential of pathogens transmission across continents. This study aimed to perform a diagnostic screening of intended bacteria associated with RTIs among Malaysian Hajj pilgrims by using a newly developed PCR assay. Expectorated sputum specimens (n = 202) and sociodemographic characteristics of the returning Hajj pilgrims were collected upon arrival in Kelantan, Malaysia. Diagnostic screening of bacterial respiratory pathogens was performed using a thermostabilized multiplex PCR assay in parallel with the sputum culture. Of the six intended bacteria: Haemophilus influenzae, Klebsiella pneumoniae, Mycobacterium tuberculosis, Pseudomonas aeruginosa, Staphylococcus aureus and Streptococcus pneumoniae, the sputum specimens were found positive for H. influenzae (n = 139), K. pneumoniae (n = 20), and S. pneumoniae (n = 19) by the multiplex PCR assay. The sensitivity, specificity, positive- and negative predictive values (PPV and NPV) of this assay were 100% (95% confidence interval (CI): 97.85% to 100.00%), 92.23% (95% CI: 85.27% to 96.59%), 95.51% (95% CI: 91.61% to 97.64%) and 100.00%, respectively. The accuracy of this assay was 97.07% (95% CI: 94.31% to 98.73%). Overall, H. influenzae was found to be the predominant organism in the pilgrims' sputa by both molecular and microbial culture methods. The multiplex PCR assay would enable a simple, faster and reliable means for the massive screening of intended bacteria compared to the sputum culture, especially during the Hajj pilgrimage.

Real-time loop-mediated isothermal amplification assay for rapid detection of human papillomavirus 16 in oral squamous cell carcinoma.

OBJECTIVE: The present study established a real-time loop-mediated isothermal amplification (qLAMP) for rapid detection of human papillomavirus subtype 16 (HPV-16) in oral squamous cell carcinoma (OSCC). METHODS: The qLAMP assay was optimized targeting the HPV-16 E7 gene. The analytical sensitivity and specificity of the assay were determined using HPV-18 (ATCC® 45152D™), HPV-35 (ATCC® 40330™), HPV-43 (ATCC® 40338™) and HPV-56 (ATCC® 40549™) viral strains and oral bacteria. HPV-16 standard curve was constructed for determination of HPV-16 viral load. The diagnostic performance of the assay was evaluated from 63 OSCC patients comprising 63 tissue, 13 saliva and 49 blood samples, in comparison with p16 immunohistochemistry (IHC), in-house PCR and nested PCR assays. RESULTS: The detection limit of developed LAMP and PCR assays was 4.68 × 101 and 4.68 × 103 copies/µl, respectively. qLAMP assay enabled detection of positive results as early as 23 min at 67 °C. This assay can detect HPV-16 positivity in 23 % (3/13) saliva and 4.8 % (3/63) tissue samples with the viral load ranging from 4.68 × 101 to 4.68 × 104 copies/µl. HPV-16 positivity was not detected in all the blood samples. The sensitivity and specificity of qLAMP were 100 % in comparison with that of p16 IHC and nested PCR. CONCLUSION: This study reports for the first time on the use of qLAMP assay for detection of HPV-16 in OSCC in both tissue and saliva as the sample matrix which holds promise in improving the diagnostic application owing to its rapidity, simplicity, high sensitivity and specificity.

Thermostable Heptaplex PCR Assay for the Detection of Six Respiratory Bacterial Pathogens.

A thermostabilized, multiplex polymerase chain reaction (mPCR) assay was developed in this study for the detection of six respiratory bacterial pathogens. Specific primers were designed for an internal amplification control (IAC) and six target sequences from Klebsiella pneumoniae, Staphylococcus aureus, Streptococcus pneumoniae, Pseudomonas aeruginosa, Mycobacterium tuberculosis, and Haemophilus influenzae. The resultant seven-band positive amplification control (PAC) of this heptaplex PCR assay corresponded to 105 base pairs (bp) of IAC, 202 bp of K. pneumoniae, 293 bp of S. aureus, 349 bp of S. pneumoniae, 444 bp of P. aeruginosa, 505 bp of M. tuberculosis, and 582 bp of H. influenzae. Results found that 6% (w/v) of the stabilizer was optimum to preserve the functional conformation of Taq DNA polymerase enzyme. This assay was stable at ambient temperature for at least 6 months. The sensitivity and specificity of this assay were both 100% when testing on the intended target organisms (n = 119) and non-intended species (n = 57). The mPCR assay developed in this study enabled accurate, rapid, and simple detection of six respiratory bacteria.

Cytokine (IL-10, IL-6, TNF-α and TGF-ß1) Gene Polymorphisms in Chronic Hepatitis C Virus Infection among Malay Male Drug Abusers.

Cytokines play an important role in modulating inflammation during viral infection, including hepatitis C virus (HCV) infection. Genetic polymorphisms of cytokines can alter the immune response against this infection. The objective of this study was to investigate the possible association between chronic hepatitis C virus infection susceptibility and cytokine gene polymorphism for interleukin-10 (IL-10) rs1800896 and rs1800871, interleukin 6 (IL-6) rs1800795, TNF-α rs1800629, and TGF-ß1 rs1800471 in Malay male drug abusers. The study was conducted on 76 HCV-positive (HP) male drug abusers and 40 controls (HCV-negative male drug abusers). We found that there were significant differences in the frequencies of genotype for IL-10 rs1800871 (p = 0.0386) and at the allelic level for IL-10 rs1800896 A versus G allele (p = 0.0142) between the HP group and the control group. However, there were no significant differences in gene polymorphism in interleukin 6 rs1800795, TNF-α rs1800629 and TGF-ß1 rs1800471. These findings suggest significant associations between gene polymorphism for IL-10 rs1800871, IL-10 rs1800896 (at the allelic level) and susceptibility to HCV infection among Malay male drug abusers.

Aptasensor for the Detection of Mycobacterium tuberculosis in Sputum Utilising CFP10-ESAT6 Protein as a Selective Biomarker.

A portable electrochemical aptamer-antibody based sandwich biosensor has been designed and successfully developed using an aptamer bioreceptor immobilized onto a screen-printed electrode surface for Mycobacterium tuberculosis (M. tuberculosis) detection in clinical sputum samples. In the sensing strategy, a CFP10-ESAT6 binding aptamer was immobilized onto a graphene/polyaniline (GP/PANI)-modified gold working electrode by covalent binding via glutaraldehyde linkage. Upon interaction with the CFP10-ESAT6 antigen target, the aptamer will capture the target where the nano-labelled Fe3O4/Au MNPs conjugated antibody is used to complete the sandwich format and enhance the signal produced from the aptamer-antigen interaction. Using this strategy, the detection of CFP10-ESAT6 antigen was conducted in the concentration range of 5 to 500 ng/mL. From the analysis, the detection limit was found to be 1.5 ng/mL, thereby demonstrating the efficiency of the aptamer as a bioreceptor. The specificity study was carried out using bovine serum albumin (BSA), MPT64, and human serum, and the result demonstrated good specificity that is 7% higher than the antibody-antigen interaction reported in a previous study. The fabricated aptasensor for M. tuberculosis analysis shows good reproducibility with an relative standard deviation (RSD) of 2.5%. Further analysis of M. tuberculosis in sputum samples have shown good correlation with the culture method with 100% specificity and sensitivity, thus making the aptasensor a promising candidate for M. tuberculosis detection considering its high specificity and sensitivity with clinical samples.

Dataset of complete genome assembly and analysis of mycobacterium tuberculosis strain SIT745/EAI1-MYS.

In this dataset, we report the genome assembly and data analysis of Mycobacterium tuberculosis strain SIT745/EAI1-MYS. Previously, this strain was isolated from a Malaysian patient with extra-pulmonary tuberculosis, and identification of this strain is done by spoligotype patterns with fifteen known Shared International Type (SITs). Further analysis showed that this strain has a remarkable phylogeographical specificity for Malaysia. Based on the National Center for Biotechnology Information (NCBI) nucleotide database information, the complete genome consists of 150 contigs with various sequence lengths and was not assembled. In this assembly, the aforementioned contigs along with reference sequence from Mycobacterium tuberculosis strain H37Rv and Mycobacterium bovis strain AF2122/97 was used for gap closures, were assembled into a single circular chromosome length of approximately 4.42 Mega bases (Mb) with an average GC content of 65.6%. The single circular chromosome was shown to contain 4,009 protein-coding sequences, 3 ribosomal RNAs, 45 transfer RNAs, and 12 superclasses distributed with 277 subsystems which constitute nearly 1900 genes, respectively. The genome information will provide fundamental knowledge of this organism as well as insight for understanding genomic and proteomic profiling, phylogenetic relationship.

Detection of human papillomavirus in oropharyngeal squamous cell carcinoma.

Worldwide there has been a significant increase in the incidence of oropharyngeal squamous cell carcinoma (OPSCC) etiologically attributed to oncogenic human papillomavirus (HPV). Reliable and accurate identification and detection tools are important as the incidence of HPV-related cancer is on the rise. Several HPV detection methods for OPSCC have been developed and each has its own advantages and disadvantages in regard to sensitivity, specificity, and technical difficulty. This review summarizes our current knowledge of molecular methods for detecting HPV in OPSCC, including HPV DNA/RNA polymerase chain reaction (PCR), loop-mediated isothermal amplification (LAMP), p16 immunohistochemistry (IHC), and DNA/RNA in situ hybridization (ISH) assays. This summary may facilitate the selection of a suitable method for detecting HPV infection, and therefore may help in the early diagnosis of HPV-related carcinoma to reduce its mortality, incidence, and morbidity.

Microbial biodiversity in the throats of pulmonary tuberculosis patients and tuberculin skin test (TST) positive and negative healthy individuals in Malaysia.

The purpose of this study was to investigate the composition of throat microbiota in pulmonary tuberculosis patients (PTB) in comparison to healthy tuberculin skin test positive (TSTp) and negative (TSTn) individuals. Throat swabs samples were collected, and the microbiota was characterized. Richer operational taxonomic units (OTUs) were present in PTB group, compared to TSTp and TSTn. Regarding alpha diversity analysis there was a higher community diversity in TSTn compared to TSTp. Beta diversity analysis showed different species composition in TSTp compared to TSTn and PTB. There was higher presence of Firmicutes in PTB and TSTn compared to TSTp group at phylum level. At the genus level, Leuconostoc and Enterococcus were higher in TSTn compared to TSTp and Pediococcus, Chryseobacterium, Bifidobacterium, Butyrivibrio, and Bulleidia were higher in PTB compared to TSTn. Streptococcus was higher in TSTn compared to PTB and Lactobacillus in PTB compared to TSTp. At species level, Streptococcus sobrinus and Bulleidia moorei were higher in PTB compared to TSTn individuals, while Lactobacillus salivarius was higher in PTB compared to TSTp. The differences in the microbiome composition could influence the resistance/susceptibility to Mtb infection.

High frequency of Haemophilus influenzae associated with respiratory tract infections among Malaysian Hajj pilgrims.

BACKGROUND: Overcrowding during the annual Hajj pilgrimage has been known to increase the risk of infectious diseases transmission. Despite the high prevalence of respiratory illness among Malaysian Hajj pilgrims, knowledge about the etiologic pathogens is yet very limited. Thus, this study aimed to determine the spectrum of bacterial respiratory pathogens among the Hajj pilgrims returning to Malaysia in year 2016. METHODS: Expectorated sputum specimens were collected from the Hajj pilgrims with symptomatic respiratory tract infections (RTIs). Subsequently, the bacterial pathogens were identified using the standard bacteriological culture method and Vitek II system. RESULTS: This study indicated that 255 (87.33%) out of 292 cultured sputa were positive with at least one potential pathogenic bacteria. Out of 345 total bacterial isolates, 60% (n=207) were Haemophilus influenzae, which was associated with both single bacterium infection (132/173, 76.3%) and multiple bacterial infections (75/82, 91.5%). The other bacterial isolates included; Klebsiella pneumoniae (n=37, 10.7%), Moraxella catarrhalis (n=27, 7.8%), Haemophilus parainfluenzae (n=25, 7.2%), Streptococcus group G (n=18, 5.2%), Klebsiella spesies (n=16, 4.6%), Streptococcus pneumoniae (n=11, 3.2%) and few other organisms. CONCLUSION: High frequency of H. influenzae was isolated from Malaysian Hajj pilgrims, especially those with respiratory symptoms. Further study should evaluate the actual pathogenicity of the organism and the interactions between the respiratory microbiota towards developing effective prevention strategies of RTIs among the local pilgrims.

Immune TB Antibody Phage Display Library as a Tool To Study B Cell Immunity in TB Infections.

B cells and in particular antibodies has always played second fiddle to cellular immunity in regard to tuberculosis (TB). However, recent studies has helped position humoral immunity especially antibodies back into the foray in relation to TB immunity. Therefore, the ability to correlate the natural antibody responses of infected individuals toward TB antigens would help strengthen this concept. Phage display is an intriguing approach that can be utilized to study antibody-mediated responses against a particular infection via harvesting the B cell repertoire from infected individuals. The development of disease-specific antibody libraries or immune libraries is useful to better understand antibody-mediated immune responses against specific disease antigens. This study describes the generation of an immune single-chain variable fragment (scFv) library derived from TB-infected individuals. The immune library with an estimated diversity of 109 independent clones was then applied for the identification of monoclonal antibodies against Mycobacterium tuberculosis α-crystalline as a model antigen. Biopanning of the library isolated three monoclonal antibodies with unique gene usage. This strengthens the role of antibodies in TB immunity in addition to the role played by cellular immunity. The developed library can be applied against other TB antigens and aid antibody-derived TB immunity studies in the future.

High burden of Carbapenem-resistant Enterobacteriaceae (CRE) fecal carriage at a teaching hospital: cost-effectiveness of screening in low-resource setting.

BACKGROUND: Infections by multidrug-resistant gram-negative bacteria (MDR-GNB) have been continuously growing and pose challenge to health institution globally. Carbapenem-resistant Enterobacteriacea (CRE) was identified as one of the MDR-GNB which has limited treatment options and higher mortality compared to those of sensitive strains. We report an increased burden of CRE fecal carriage at a hospital in the North-eastern region of Malaysia. METHODS: A retrospective descriptive study from August 2013 to December 2015 was conducted in the Medical Microbiology & Parasitology laboratory of Hospital Universiti Sains Malaysia, which is a tertiary teaching hospital with more than 700 beds. This hospital treats patients with various medical and surgical conditions. Suspected CRE from any clinical specimens received by the laboratory was identified and confirmed using standard protocols. Polymerase chain reaction (PCR) assay was performed to determine the genotype. RESULTS: Altogether, 8306 Enterobacteriaceae was isolated from various clinical specimens during the study period and 477/8306 (5.74%) were CRE. Majority of the isolated CRE were Klebsiella [408/477, (85.5%)], of which Klebsiella pneumoniae was the predominant species, 388/408 (95%). CRE were mainly isolated from rectal swab (screening), 235/477 (49.3%); urine, 76/477 (15.9%); blood, 46/477 (9.6%) and about 7.1% from tracheal aspirate. One hundred and thirty-six isolates were subjected to genotype determination and., 112/136 (82.4%) showed positive detection of New Delhi metallo-ß-lactamase 1 (NDM-1) gene (blaNDM1). CONCLUSION: The study noted a high numbers of CRE isolated especially from rectal swabs. Active screening results in significant cost pressures and therefore should be revisited and revised, especially in low resource settings.

A Thermostabilized, One-Step PCR Assay for Simultaneous Detection of Klebsiella pneumoniae and Haemophilus influenzae.

Klebsiella pneumoniae and Haemophilus influenzae are two common pathogens associated with respiratory tract infections. The identification of these pathogens using conventional molecular diagnostic tests requires trained personnel, cold-chain transportation, and storage-dependance, which does not render them user-friendly. The aim of this study was to develop a thermostabilized, cold-chain-free, one-step multiplex PCR for simultaneous detection of K. pneumoniae and H. influenzae. The multiplex PCR assay was designed to amplify the php gene of K. pneumoniae (202 bp) and p6 gene of H. influenzae (582 bp). In addition, the specific primer to amplify glm gene of Helicobacter pylori (105 bp) was included as an internal amplification control. Subsequently, the designed primers and all PCR reagents were thermostabilized by lyophilization. The stability of the thermostabilized PCR was evaluated using the Q10 method. The sensitivity and specificity of performances for thermostabilized PCR were evaluated using 127 clinical isolates and were found to be 100% sensitive and specific. The thermostabilized PCR mix was found to be stable for 30 days and the Q10 accelerated stability was found to be 3.02 months. A cold-chain-free, PCR assay for easy, rapid, and simultaneous detection of K. pneumoniae and H. influenzae was successfully developed in this study.

The prevalence and preventive measures of the respiratory illness among Malaysian pilgrims in 2013 Hajj season.

BACKGROUND: Respiratory illness continues to exert a burden on hajj pilgrims in Makkah. The purpose of this study is to determine the prevalence of respiratory illness and its associated factors among Malaysian hajj pilgrims in 2013 and to describe its preventive measures. METHODS: A cross-sectional study was conducted in Makkah and Malaysia during the 2013 hajj season. A self-administered proforma on social demographics, previous experience of hajj or umrah, smoking habits, co-morbid illness and practices of preventive measures against respiratory illness were obtained. RESULTS: A total of 468 proforma were analysed. The prevalence of the respiratory illness was 93.4% with a subset of 78.2% fulfilled the criteria for influenza-like illness (ILI). Most of them (77.8%) had a respiratory illness of <2 weeks duration. Approximately 61.8% were administered antibiotics but only 2.1% of them had been hospitalized. Most of them acquired the infection after a brief stay at Arafat (81.2%). Vaccination coverages for influenza virus and pneumococcal disease were quite high, 65.2% and 59.4%, respectively. For other preventive measures practices, only 31.8% of them practiced good hand hygiene, ∼82.9% of pilgrims used surgical face masks, N95 face masks, dry towels, wet towels or veils as their face masks. Nearly one-half of the respondents (44.4%) took vitamins as their food supplement. Malaysian hajj pilgrims with previous experience of hajj (OR 0.24; 95% CI 0.10-0.56) or umrah (OR 0.19; 95% CI 0.07-0.52) and those who have practiced good hand hygiene (OR 0.35; 95% CI 0.16-0.79) were found to be significantly associated with lower risk of having respiratory illness. Otherwise, pilgrims who had contact with those with respiratory illness (OR 2.61; 95% CI 1.12-6.09) was associated with higher risk. CONCLUSIONS: The prevalence of respiratory illness remains high among Malaysian hajj pilgrims despite having some practices of preventive measures. All preventive measures which include hand hygiene, wearing face masks and influenza vaccination must be practiced together as bundle of care to reduce respiratory illness effectively.