Cases of autochthonous infections of dengue virus type 1 (DENV-1) were detected in Japan after a 70-year period devoid of dengue outbreaks. We previously showed that E gene sequences are identical in 11 of the 12 DENV-1 strains autochthonous to Japan. However, the E sequence represents only 14% of the DENV-1 genome. In the present study, we have sequenced the entire genome of 6 autochthonous DENV-1 strains that were isolated from patients during the 2014 outbreak. Sequencing of 5 Yoyogi group strains with identical E sequences and 1 Shizuoka strain with a different E sequence revealed that the first Yoyogi group strain differed from the Shizuoka strain by 18 amino acid residues. Furthermore, 2 Yoyogi group strains had different genomic sequences while the other 3 had identical genomes. Phylogenetic analyses indicated that the Hyogo strain, a Yoyogi group strain, was the first to diverge from the other 4 Yoyogi group strains. The E gene sequence of the Yoyogi group strains exhibits the highest homology to those of the strains isolated in Malaysia and Singapore between 2013 and 2014. The patient infected with the Hyogo strain visited Malaysia before the onset of dengue fever, suggesting that this was a case of dengue infection imported from Malaysia.
The genes for Nipah virus (NiV) proteins were amplified from viral RNA, cloned into the plasmid pTriEx-3 Hygro, expressed, and purified using immobilized metal affinity chromatography. The recombinant N, F, and G NiV proteins (rNiV-N, rNiV-F, and rNiV-G), were successfully expressed in Escherichia coli and purified with a yield of 4, 16, and 4 mg/L, respectively. All 3 recombinant viral proteins reacted with all 19 samples of NiV-positive human sera. The rNiV-N and rNiV-G proteins were the most immunogenic. The recombinant viral proteins did not react with any of the 12 NiV-negative sera. However, serum from a patient with a late-onset relapsing NiV infection complication was found to be primarily reactive to rNiV-G only. Additionally, there is a distinctive variation in the profile of antigen-reactive bands between the sample from a case of relapsing NiV encephalitis and that of acute NiV infection. The overall findings of this study suggest that the recombinant viral proteins have the potential to be developed further for use in the detection of NiV infection, and continuous biosurveillance of NiV infection in resource-limited settings.
The prevalence of ceftriaxone resistance and the associated genes encoding extended-spectrum β-lactamase (ESBL) was determined in 149 non-duplicate non-typhoidal Salmonella isolated in 2008-2009 from patients in a tertiary care hospital in Kuala Lumpur, Malaysia. The resistance rate to ceftriaxone was 2.7% (2/74) in 2008, 4.0% (3/75) in 2009, and 3.4% (5/149) overall. CTX-M ESBL genes were detected in 2 of the 5 ceftriaxone-resistant isolates. The prevalence of ceftriaxone resistance, although low, is a concern because it limits therapeutic options. Continued surveillance of ceftriaxone resistance is important to monitor its trends.
Restriction enzymes SpeI and XbaI were used in a pulsed-field gel electrophoresis (PFGE) study for molecular characterization of 146 clinical Burkholderia pseudomallei isolates. The PFGE parameters were optimized to enable comparable, reproducible, and robust results. The optimized parameters for both SpeI and XbaI restriction enzymes used in this study were 200 V and a pulse time of 5 to 65 s for a 28-h runtime. Using SpeI, 9 different clusters were identified, whereas 6 clusters were identified by XbaI digestion, which exhibited 85% similarity to SpeI. SpeI (discrimination index [D]=0.854) showed higher discriminatory power than XbaI did (D=0.464).
The resistance phenotypes and genomic diversity of 185 Acinetobacter baumannii isolates obtained from the intensive care unit (ICU) of a local teaching hospital in Kuala Lumpur from 2006 to 2009 were determined using antimicrobial susceptibility testing and pulsed-field gel electrophoresis (PFGE). Antibiogram analyses showed that the isolates were fully resistant to β-lactam antimicrobials and had high resistance rates to the other antimicrobial agents tested. However, the isolates were susceptible to polymyxin B. Resistance to cefoperazone/sulbactam was only detected in strains isolated from 2007 to 2009. Some environmental isolates and an isolate from the hands of a healthcare worker (HCW) had identical resistance profiles and PFGE profiles that were closely related to patient isolates. Cluster analyses based on the PFGE profiles showed there was a persistent clone of endemic isolates in the ICU environment. The transmission route from HCWs to fomites to patients, which caused a long-term infection in the ICU of the University Malaya Medical Centre, was observed in this study. These data provide a better understanding of A. baumannii epidemiology within the hospital and the possible transmission routes. Knowledge of changes in the resistance rates of A. baumannii in our local hospital will improve antimicrobial therapy.
This study was aimed at determining the molecular epidemiology of rabies virus (RABV) circulating in Vietnam. Intra vitam samples (saliva and cerebrospinal fluid) were collected from 31 patients who were believed to have rabies and were admitted to hospitals in northern provinces of Vietnam. Brain samples were collected from 176 sick or furious rabid dogs from all over the country. The human and canine samples were subjected to reverse transcription-polymerase chain reaction analysis. The findings showed that 23 patients tested positive for RABV. Interestingly, 5 rabies patients did not have any history of dog or cat bites, but they had an experience of butchering dogs or cats, or consuming their meat. RABV was also detected in 2 of the 100 sick dogs from slaughterhouses. Molecular epidemiological analysis of 27 RABV strains showed that these viruses could be classified into two groups. The RABVs classified into Group 1 were distributed throughout Vietnam and had sequence similarity with the strains from China, Thailand, Malaysia, and the Philippines. However, the RABVs classified into Group 2 were only found in the northern provinces of Vietnam and showed high sequence similarity with the strain from southern China. This finding suggested the recent influx of Group 2 RABVs between Vietnam and China across the border. Although the incidence of rabies due to circulating RABVs in slaughterhouses is less common than that due to dog bite, the national program for rabies control and prevention in Vietnam should include monitoring of the health of dogs meant for human consumption and vaccination for workers at dog slaughterhouses. Further, monitoring of and research on the circulating RABVs in dog markets may help to determine the cause of rabies and control the spread of rabies in slaughterhouses in Vietnam.
We investigated the prevalence of non-typhoidal Salmonella (NTS) with "reduced susceptibility to ciprofloxacin" (RS-Cip) (minimum inhibitory concentration [MIC], 0.12-1.0 μg/mL) as well as their resistance genes in 75 NTS isolates (53 from stool, 21 from blood, and 1 from urine) from patients at a tertiary care Malaysian hospital between January and December 2009. RS-Cip was detected in 24/75 (32.0%) isolates. Using the ciprofloxacin MIC interpretive criteria for Salmonella in the Clinical and Laboratory Standards Institute 2013 guidelines, 51/75 (68.0%) isolates were found to be sensitive, 22/75 (29.3%) were intermediate, and 2/75 (2.7%) were resistant to ciprofloxacin. The 24 isolates that were intermediate or resistant to ciprofloxacin were the same isolates categorized as having RS-Cip. Among the 23 tested isolates with RS-Cip, the qnrS gene was detected in 17/23 (73.9%) and single gyrA mutations were detected in 6/23 (26.1%) (Asp87Tyr [n = 3], Asp87Asn [n = 2], and Ser83Phe [n = 1]). A parC (Thr57Ser) mutation was detected in 13/23 (56.5%) isolates, coexisting with either a qnrS gene or a gyrA mutation. The high incidence of the qnrS gene among isolates with RS-Cip needs to be monitored because qnr genes can spread via plasmids and aid in the emergence of increased resistance levels.
Group B Streptococcus (GBS) infection was studied in 49 patients collected at convenience (convenience sampling), excluding infants and women with genital tract- and pregnancy-related isolates, according to the availability of stocked isolates and easy accessibility to epidemiological data. The data were examined both prospectively and retrospectively from 2003-2005 at a tertiary-level multidisciplinary hospital in Kuala Lumpur, Malaysia. Skin and soft-tissue infections in 35 patients (71.4%) were the most common clinical presentation, while diabetes mellitus was the most common underlying condition (35 patients, 71.4%). All GBS isolates were sensitive to penicillin, and most isolates tested were sensitive to erythromycin (97.7%). Serotyping of 45 GBS isolates using a commercial serotyping kit revealed that the most common serotype was Ia (22.2%), followed by VI (17.8%), III and V (13.3% each). Others included Ib, II, IV, VIII, and VII; 13.3% were nontypeable. The findings of this pilot study are limited by the small sample size, the sampling method and the possibility that the cases are not wholly representative of the University Malaya Medical Centre population. Further studies from our hospital with larger numbers and using probabilistic sampling techniques are required to confirm the relatively high occurrence of serotype VI (the second most common serotype) in the population studied.
The genetic diversity and antimicrobial resistance rates of clinical Salmonella isolates (2007-2008) at the University of Malaya Medical Centre, Kuala Lumpur, were investigated and the genetic diversity of the isolates was determined by pulsed-field gel electrophoresis (PFGE) and repetitive extragenic palindromic (REP)-PCR. XbaI-PFGE analysis generated 57 profiles (Dice coefficient, F=0.08-1.00), whereas REP-PCR using the REP primer generated only 35 (F=0.34-1.00). PFGE was therefore the more discriminative and reproducible method for assessing the genetic diversity of salmonellae. The antibiograms of 78 Salmonella isolates were assessed against 19 antimicrobials using the disk diffusion method. Twenty serotypes were identified, with the most common being S. Enteritidis (18%) followed by S. Typhimurium (14%), S. Paratyphi B var Java (9%), S. Weltevreden (9%), and S. Corvallis (9%). A total of 38 resistant profiles were defined, with 53.8% of the isolates being resistant to three or more antimicrobials. The highest resistance rates were observed for cephalothin (55.1%), tetracycline (47.4%), and nalidixic acid (35.9%). The presence of multidrug-resistant Salmonella strains is a cause for concern as it may limit the treatment of severe salmonellosis. One multidrug-resistant S. Enteritidis strain was a putative extended-spectrum beta-lactamase producer, based on a double disk diffusion analysis, and was resistant to ceftriaxone (MIC>32 microg/mL). The data generated by this study will contribute towards epidemiological monitoring and investigations of Salmonella infections in Malaysia.
Panton-Valentine leukocidin (PVL) is a cytotoxin which causes leukocyte destruction and tissue necrosis. Although it is produced by fewer than 5% of Staphylococcus aureus strains, PVL-producing S. aureus is emerging as a serious problem worldwide. There has been a marked increase in the incidence of necrotizing lung infections with a very high mortality associated with these strains. This report describes a fatal case of hospital-acquired necrotizing pneumonia caused by PVL-positive methicillin-susceptible S. aureus in a patient with a brain tumor.
Mupirocin is used topically to treat skin infection caused by methicillin-resistant Staphylococcus aureus (MRSA). One hundred eighty-eight strains (isolated in 2003, 2004, 2007, and 2008) were tested for mupirocin susceptibility using disk diffusion method and minimum inhibitory concentration (MIC). Mupirocin resistance was detected in 10 (5%) strains with 2 of them showing MIC of 256 mg/l. PCR detection using gene-specific primers showed that all 10 mupirocin-resistant strains harbored ileS2 gene whereas mupA gene was detected in 2 mupirocin-resistant strains with MIC of 256 mg/l. Amplification of agr grouping and SCCmec typing showed that all 10 strains were agr group I and SCCmec type III. Sequence analysis of region X of the spa gene yielded 4 distinct spa types (t037, t363, t421, and t6405) which were clonally related. In conclusion, the rate of mupirocin resistance in Malaysia is still low but is much higher than previous reports in Malaysia.
Salmonella enterica serovar Paratyphi A is a causative agent of paratyphoid fever. The clinical syndrome caused by paratyphoid fever overlaps with other febrile illnesses and cannot be distinguished from typhoid fever. Conventional methods used for diagnosis are time consuming, costly, and labor-intensive. We evaluated the specificity, sensitivity, and application of a multiplex polymerase chain reaction (PCR) previously developed by the method (Ou, H.Y., Teh, C.S.J., Thong, K.L., et al., J. Mol. Diagn., 9, 624-630, 2007) using 6 S. Paratyphi A, 22 S. Typhi, and 85 other Salmonella serovars as well as 36 non-Salmonella strains. The detection limit of the multiplex PCR was 4 x 10(4) cfu ml(-1). In a blind test of the other 50 strains, this multiplex PCR correctly identified the only S. Paratyphi A in the panel of strains. The sensitivity of this PCR using spiked blood and stool samples was 1 x 10(5) cfu ml(-1) and 2 x 10(5) cfu ml(-1), respectively, but increased to 1 x 10(4) cfu ml(-1) and 2 x 10(3) cfu ml(-1) after 5-h enrichment. We believe that this multiplex PCR is a promising technique for the specific and sensitive detection of S. Paratyphi A in clinical, environmental, and food samples.
Determining the local circulating strain of influenza is essential to prevent and control epidemics. In the years 2004 and 2005, the National Influenza Center of Thailand received 3,854 and 3,834 specimens, respectively, from patients throughout the country, including submissions from 4 established influenza surveillance sentinel sites. In 2004, of 539 influenza-positive specimens, 461 were positive for influenza A and 78 were positive for influenza B by isolation. Influenza A subtyping revealed that 249, 197, and 15 isolates were H1N1, H3N2, and H5N1, respectively. In 2005, of 748 influenza-positive specimens, 492 were influenza A and the remaining 256 were influenza B. The results of influenza A subtyping indicated that 55, 437, and 5 isolates were H1N1, H3N2, and H5N1. All isolated strains of subtype H1N1 were A/New Caledonia/20/99-like. The isolated strains of H3N2 were A/Fujian/411/2002-like in the first half of the year 2004, while those in the latter half of 2004 gradually drifted to a mixture of A/Wellington/1/2004-like, A/California/7/2004-like, and A/Wisconsin/67/2005-like, and this mixture continued through the end of 2005. The influenza B strains were B/Sichuan/379/99-like, B/Hong Kong/330/2001-like, B/Shanghai/361/2002-like and B/Malaysia/2506/2004-like. The strains circulating in the years 2004 and 2005 were antigenically similar to the vaccine formulas recommended in the same period by WHO. Our results underscore that local influenza surveillance plays an important role in responding to epidemics and potential pandemics.
The purpose of this study was to examine the extent of dengue infection in Brunei and to determine the predominant serotype circulating in the country. The study generated useful epidemiological data on dengue infection in Brunei. A total of 271 samples from patients suspected of having dengue infections were selected and analyzed. All patients were seen in clinics and hospitals in Brunei. The samples were collected from April 2005 to April 2006 and transported to the WHO Collaborating Centre for Arbovirus Reference and Research, University of Malaya, Malaysia. The following tests were used to achieve the objectives: in-house IgM-capture enzyme-linked immunosorbent assay, virus isolation in mosquito albopictus cell line (C6/36), and viral RNA detection and serotyping by reverse transcriptase-polymerase chain reaction (RT-PCR). The results show that 45 people were positive for dengue-specific IgM (27 males and 18 females), while RT-PCR detected dengue viral RNA in 12 patients, 3 identified as DEN-1 and 9 as DEN-2. Dengue virus was isolated from 6 patients using the C6/36 cell line; 3 were DEN-2 isolates and 3 were DEN-1 isolates. These data show that dengue virus is circulating in Brunei and the predominant infecting serotype for that period was DEN-2 followed by DEN-1. This study is the first to report the detection and isolation of dengue virus from Brunei using RT-PCR and culture in the C6/36 albopictus mosquito cell line.
Fluoxetine (FLX), a P-glycoprotein inhibitor with antimalarial activity, is promising candidate for reversing chloroquine/mefloquine (CQ/MQ) resistance. The Dd2 strain of CQ- and MQ-resistant Plasmodium falciparum was synchronized and assayed with various concentrations of CQ/MQ individually and in combination with FLX or verapamil (VPL). Our results indicated the 50% inhibitory concentration values of CQ and MQ were greatly lowered when FLX was used simultaneously. Isobolograms indicated that CQ-FLX combinations are more synergistic (mean fractional inhibitory concentration [FIC] index 0.55) than MQ-FLX (mean FIC index 0.64), and their synergistic effect was better than or at par with CQ-VPL (mean FIC index 0.88) and MQ-VPL (mean FIC index 0.60) combinations. We conclude that the FLX potentiates the CQ and MQ response on multidrug-resistant P. falciparum at clinically achievable concentrations.
Pantoea infections are uncommon in humans. Most reports have involved adults or children after thorn injuries. There are only a few reports of systemic infections with Pantoea. This is the first report of the clinical picture of systemic Pantoea spp. infection in neonates as observed during an outbreak in a neonatal intensive care unit caused by infected parenteral nutrition solutions. Even though detected early, the infections had a fulminant course, causing septicemic shock and respiratory failure. Pulmonary disease was prominent and presented mainly as pulmonary hemorrhage and adult respiratory distress syndrome. The organism was sensitive to most antibiotics used in neonatal intensive care units, but the clinical response to antibiotic therapy was poor. The fatality rate was very high: 7 out of 8 infected infants succumbed to the infection (87.5%).
Human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome (AIDS) presents a serious healthcare threat to young individuals in Malaysia and worldwide. This study aimed to identify trends in HIV-related risk behaviors among recognized high-risk groups and to estimate HIV transmission up to the year 2015. Data and necessary information were obtained from the Ministry of Health Malaysia, published reports from the World Health Organization and United Nations Program on HIV/AIDS, and other articles. The Estimation and Projection Package was used to estimate HIV transmission. The results of the present study revealed that within the high-risk groups, intravenous drug users (IDUs) had the highest prevalence rate of HIV transmission, followed by patients with sexually transmitted infections (STIs), female sex workers (SWs), and men who have sex with men (MSM). Within these at-risk populations, patients with STIs have the highest prevalence of HIV, followed by IDUs, MSM, and SWs. If the transmission rate continues to increase, the situation will worsen; therefore, there is an urgent need for a comprehensive prevention program to control HIV transmission in Malaysia.