Human papillomaviruses (HPVs) are the causative agent of cervical cancer, and among approximately 15 high-risk genotypes, HPV16 accounts for more than half the cases of cervical cancer worldwide. Recent progress in determining HPV genomic sequences from clinical samples has revealed a wide variety in HPV16 genome sequences, and has allowed for comprehensive classification of intratype HPV16 variants. These consist of four variant lineages containing nucleotide variations in 1.0%–10.0% of the complete viral genome sequence. Epidemiological data suggest that the non-European–Asian lineages of HPV16 entail a higher risk of progression to invasive cervical cancer than the European–Asian lineage. Deep sequencing analysis has recently demonstrated that HPV16 genome sequences are highly homogeneous in individual clinical specimens compared with those of RNA viruses. However, an extremely sensitive PCR method, differential DNA denaturation PCR, has detected hypermutations from C to T or G to A in the E2 gene and the long control region of the HPV16 genome, which suggests the involvement of cellular apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like (APOBEC) proteins in this hypermutation. The quasispecies status of the HPV16 genome in the infected cervix may affect the development of cervical cancer and warrants further investigation.

View full abstract

Infection with different strains of Helocobacter pylori and emergence of new genomic variants during their long-term gastric colonization are assumed to be the main reasons for eradication failure. We used genotyping and arbitrarily primed PCR fingerprinting (RAPD) to detect relatedness and genetic variations among H. pylori single isolates from each patient in Iran. Multiplex-PCR amplification of gene alleles encoding the virulence factors vacA (m/s), cagA, and iceA (A1/A2) and comparison of RAPD patterns of different singles colonies were performed for each individual patient's isolate. Results showed a high frequency of diversity among the H. pylori strains. Nearly 23% of infected patients showed a single type infection. The infection types related, unrelated, and related/unrelated were found among 25.6%, 12.8%, and 38.5% of patients, respectively. Both mixed type infections (77%) and quasispecies development (15.4%) were detected in these patients. Genotype conversion among vacA (41.6%), cagA (41.6%), and iceA (50%) alleles was observed for the strains with identical or related RAPD patterns. Coevolution of different alleles was also detected in a patient infected with strains presenting the same RAPD patterns. Collectively, results of this study revealed the occurrence of quasispecies development, mixed type infections, and changes in virulence properties of H. pylori strains among the studied patients.

View full abstract

We conducted a retrospective cohort study in the medical intensive care unit of Chaing Mai University Hospital to describe the epidemiology of ventilator-associated pneumonia (VAP) and identify prognostic indicators of 30-day VAP mortality. A total of 621 patients diagnosed with VAP between January 2005 and December 2011 were included. The overall 30-day mortality rate was 44.4%. The major causative pathogens were Acinetobacter baumannii (54.3%), Pseudomonas aeruginosa (35.2%), and methicillin-resistant Staphylococcus aureus (15.1%). Most A. baumannii (90.2%) comprised drug-resistant strains. Identified prognostic indicators were co-morbid malignancy (hazard ratio [HR] = 1.60; 95% confidence interval [CI] 1.02–2.42; P = 0.040), septic shock (HR = 2.51; 95% CI, 1.60–4.00; P < 0.001), Simplified Acute Physiology Score II >45 (HR = 1.62; 95% CI, 1.03–2.56; P = 0.041), Sequential Organ Failure Assessment score >5 (HR = 3.40; 95% CI 2.00–5.81; P < 0.001), and delayed inappropriate empirical antibiotic treatment (HR = 2.23; 95% CI, 1.12–4.45; P = 0.022). VAP was associated with high mortality. The major causative pathogen was drug-resistant A. baumannii. Therefore, early detection of VAP by surveillance in mechanically ventilated patients leading to earlier treatment may improve patient outcomes. Guidelines for prescribing appropriate empirical antibiotics to cover drug-resistant bacteria could be established using local epidemiological data.

View full abstract

We investigated a diarrhea outbreak in 2 universities to identify the etiological agent responsible, the source of infection, the mode of transmission, and the risk factors. A case-controlled study was conducted using case students and asymptomatic control students who were selected randomly and frequency-matched according to class and age, and the source of food or water intake was investigated. Of the total 22,404 students at the universities, 0.25% developed Salmonella Infections. A total of 96% (54/56) of the case students and 30% (35/117) of the control students consumed bread products provided by the same vendor (odds ratio [OR] = 63.3; 95% confidence interval [CI], 14.9–550.7). Among the students who consumed bread, 96% (52/54) of the case students and 9% (3/35) of the control students ate egg sandwiches (OR = 277.3; 95%CI, 43.9–1,750.8). Seven strains of Salmonella enteritidis and 6 strains of S. chester were isolated from the case students or food samples. Pulsed-field gel electrophoresis typing showed the same patterns. The outbreak of gastroenteritis was caused mainly by egg sandwiches contaminated with different serotypes of Salmonella.

View full abstract

In order to assess the immunity to rubella infection in Dongguan, China, we conducted a seroprevalence survey on rubella and used ELISA to measure rubella-specific IgG in serum samples. A total of 1,017 individuals aged 0–59 years were selected by multistage cluster sampling. Among them, 904 (88.9%) were seropositive for rubella. Two groups (20–29 and ≥40 years) had seropositivity rates of <90%. In comparison with participants aged ≥20 years, rubella immunization rates were higher in those aged <20 years (83.2% vs. 93.7%, respectively; χ² = 28.063, P < 0.001). Among women aged 20–29 years, only 63.8% had antibodies above the protective level. Multivariate analysis revealed that only sex and age were significantly associated with rubella-protective antibody levels. Our results suggest that in the study area, women of childbearing age had a greater serological susceptibility to rubella. Additional vaccinations for rubella of susceptible young adults should be considered, particularly in women of childbearing age.

View full abstract

We evaluated the effects of qnrA on survival of bacteria exposed to a lethal ciprofloxacin (CIP) concentration and development of quinolone resistance through the accumulation of amino acid substitutions in quinolone resistance-determining regions (QRDRs) of GyrA and ParC, targets of quinolones, in Escherichia coli. CIP-susceptible E. coli strains of different O-serotypes (O1, O6, O18, O25b, O74, and O78) were transformed by a recombinant plasmid harboring qnrA, and the parent strains and their transformants were subjected to killing curve assays and adaptation tests. In the killing curve assay at 2 × the minimum inhibitory concentration of CIP, the viable bacterial cell numbers of strains O1, O6, and O25b were maintained at 10⁵–10⁸ CFU/mL after 24-h incubation, while the remaining strains showed a 10⁵-fold reduction in viable cell numbers. In the adaptation test, a Ser83-Leu substitution in the QRDR of GyrA was identified earlier in the parent strains of O25b and O1 than in their transformants, suggesting that the acquisition of qnrA did not necessarily accelerate the rate of accumulation of amino acid substitutions in the QRDR. We confirmed that the presence of qnrA contributed to increased survival of the E. coli strains displaying certain O-serotypes. Further studies are necessary to evaluate the precise effects of qnrA on quinolone resistance acquisition by Enterobacteriaceae.

View full abstract

To develop surrogate viruses for hepatitis C virus (HCV), we previously produced recombinant vesicular stomatitis viruses (rVSVs) lacking glycoprotein G but instead expressing chimeric HCV E1/E2 fused to G. These rVSVs were not infectious in HCV-susceptible hepatoma cells. In this study, to develop an infectious surrogate HCV based on an rVSV (vesicular stomatitis virus [VSV]/HCV), we generated a novel rVSV encoding the native E1/E2 (H77 strain) and green fluorescent protein (GFP) instead of G. Here, we showed that this VSV/HCV efficiently infected human hepatoma cells, including Huh7 human hepatoma cells, expressed GFP in these cells, and propagated, but did not do so in nonsusceptible BHK-21 cells. The infectivity of VSV/HCV, measured as the number of foci of GFP-positive cells, was specifically reduced by the addition of chimpanzee anti-HCV serum, anti-E2 antibody, or anti-CD81 antibody to the cultures. When sera obtained from HCV-infected or uninfected patients were added, infection was selectively inhibited only by the sera of HCV-infected patients. These data together suggest that this infectious GFP-expressing VSV/HCV could be a useful tool for studying the mechanisms of HCV entry into cells and for assessing potential inhibitors of viral entry, including neutralizing antibodies.

View full abstract

Campylobacter-induced diarrhea is increasingly recognized worldwide. However, little information is available regarding the Campylobacter strains associated with diarrheal patients in Thailand. In this study, we attempted to isolate Campylobacter strains from diarrheal patients in Thailand and to characterize the species using a cytolethal distending toxin (cdt) gene-based C. jejuni, C. coli, and C. fetus-specific multiplex PCR assay. Campylobacter species were also confirmed using 16S rRNA gene sequencing and hipO gene detection. From 2,500 diarrheal stool specimens, 76 Campylobacter-like organisms were isolated and identified via conventional culture methods. Among these 76 organisms, 73 were identified as Campylobacter species (43 C. jejuni, 29 C. coli, and 1 C. fetus) via multiplex PCR, whereas 3 remained unidentified. Two Campylobacter-like organisms yielded 2 amplicons corresponding to cdt genes from C. jejuni and C. coli. Subsequently, C. jejuni and C. coli were re-isolated from each sample. The third isolate was identified as C. hyointestinalis via 16S rRNA gene sequencing. To our knowledge, this is the first report on the isolation of C. hyointestinalis from a diarrheal patient in Thailand. These data indicate that C. jejuni (58%) and C. coli (40%) are prevalent among diarrheal patients in Thailand.

View full abstract

The biochemical features and virulence gene profiles of 37 enterohemorrhagic Escherichia coli (EHEC) strains belonging to serogroups other than O157 and O26 (non-O157/O26 EHEC) were investigated. All strains were isolated from humans between 2002 and 2013 in the Yamaguchi Prefecture. Serogroup O111 strains were the most common, followed by O103, O121, and O145. Most strains (84%) were negative for sorbose fermentation, whereas only 1 and 2 were negative for sorbitol and rhamnose fermentation, respectively. Two strains lacked β-D-glucuronidase activity. Shiga toxin (stx) subtyping revealed 6 genotypes:stx1a (n = 20), stx1a + stx2a (n = 8), stx2a (n = 4), stx2b (n = 3), stx2a + stx2c (n = 1), and stx2a + stx2d (n = 1). Polymerase chain reaction screening of other toxin and adherence genes showed that astA, subA, and cdtB were present in 5, 2, and 2 strains, respectively. The intimin gene eae was present in 30 strains (81%). Of the 7 eae-negative strains, saa and eibG were found in 3 and 2 strains, respectively; no adherence factors were detected in the remaining 2 strains. The antimicrobial susceptibility profiles of the strains to 12 drugs were examined and 11 strains (30%) showed resistance to 1 or more drugs. Our results revealed that non-O157/O26 EHEC strains exhibit various biochemical phenotypes and carry several toxins and adherence factor genes.

View full abstract

Disease severities following dengue virus (DV) infection are the result of increased vascular permeability leading to hypovolemic shock. Matrix metalloproteinases (MMPs) are believed to play a key role in promoting such severities. A previous study reported that supernatants of DV-infected dendritic cells (DCs), which contained high levels of MMP-2 and MMP-9, induced vascular leakage in a mouse model. In the present study, we investigated whether hepatocytes (HepG2) and monocytes (U937) could be additional sources of MMPs during DV infection. HepG2 and U937 cells were exposed to DV serotype 2 strain 16681. The secretion of MMP-2 and MMP-9 was detected using gelatin zymography. We found that DV infection in the HepG2 cells promoted MMP-2 production while that in the U937 cells promoted MMP-9 production. Semi-quantitative RT-PCR results also confirmed that DV infection in the HepG2 cells up-regulated the expression of MMP-2 mRNA, whereas that in the U937 cells enhanced the expression of MMP-9 mRNA. We monitored the expression of endogenous TIMP-1 and TIMP-2. DV infection induced TIMP-1 expression in the U937 cells. However, lower expression of TIMP-2 was observed in the infected HepG2 cells. We believed that following DV infection, monocytes and hepatocytes can act as MMP-9 and MMP-2 producers, respectively. Their responses could be attributed to the disturbance of TIMP expression by DV in different cell types.

View full abstract

Yellow fever (YF), which is caused by a mosquito-borne virus, is an important viral hemorrhagic fever endemic in equatorial Africa and South America. Yellow fever virus (YFV) is the prototype of the family Flaviviridae and genus Flavivirus. The aim of this study was to determine the seroprevalence of YFV in selected health facilities in Western Kenya during the period 2010–2012. A total of 469 serum samples from febrile patients were tested for YFV antibodies using in-house IgM-capture ELISA, in-house indirect IgG ELISA, and 50% focus reduction neutralization test (FRNT₅₀). The present study did not identify any IgM ELISA-positive cases, indicating absence of recent YFV infection in the area. Twenty-eight samples (6%) tested positive for YFV IgG, because of either YFV vaccination or past exposure to various flaviviruses including YFV. Five cases were confirmed by FRNT₅₀; of these, 4 were either vaccination or natural infection during the YF outbreak in 1992–1993 or another period and 1 case was confirmed as a West Nile virus infection. Domestication and routine performance of arboviral differential diagnosis will help to address the phenomenon of pyrexia of unknown origin, contribute to arboviral research in developing countries, and enhance regular surveillance.

View full abstract

Molecular epidemiological analysis of Kudoa septempunctata isolates from 34 olive flounders associated with foodborne disease outbreaks and from 6 reference samples was performed using random amplified polymorphic DNA (RAPD) analysis. The K. septempunctata isolates analyzed in this study were divided into 8 groups. Eight isolates obtained from the large Ehime Prefecture outbreak in Japan that had occurred on October 8, 2010, were further divided into 4 groups. Eight isolates obtained from Korean samples were divided into 3 groups. These groups included isolates that had been identified from the large Ehime Prefecture outbreak. These results indicated that the Korean isolates had similar genetic backgrounds to those involved in the Ehime Prefecture outbreak. Isolates associated with outbreaks with similar dates of onset tended to be classified in the same group, suggesting that the strains involved in these incidents were genetically related. These results demonstrated that RAPD analysis is a useful molecular epidemiological analysis method for K. septempunctata.

View full abstract

The main objective of this study was to investigate the epidemiology, drug resistance and β-lactamase genotype distribution of enteropathogenic Escherichia coli (EPEC) isolated from pediatric patients with diarrhea in southern China. The prevalence of EPEC in children with diarrhea was 3.53%. The commonest serotypes were O55:K59 and O126:K71, and the typical EPEC were more prevalent than atypical EPEC (51 vs 7). Isolates from this region were most commonly found to be resistant to ampicillin and cotrimoxazole, followed by chloramphenicol, ceftriaxone, and ceftazidime. More than 96% of the strains were susceptible to cefoperazone/sulbactam and imipenem. The most common β-lactamase genotypes identified in 58 strains were bla_CTX-M-1 (60.3%), bla_TEM (56.9%), bla_CTX-M-9 (27.6%), and bla_SHV (15.5%). Among 58 isolates, 22 strains were found to harbor one β-lactamase gene, and the proportions of resistance to ampicillin, cotrimoxazole, chloramphenicol, ceftriaxone, and ceftazidime, were 81.8%, 63.6%, 40.9%, 18.2%, and 9.1%, respectively. A further 30 strains carrying multiple β-lactamase genes had increased resistance to the above antimicrobial agents (100%, 83.3%, 70.0%, 60.0%, and 30.0%, respectively). In contrast, antibiotic resistance in the last 6 strains without a detectable β-lactamase gene was substantially reduced. Drug resistance may be associated with the β-lactamase gene number, with a greater the number of β-lactamase genes resulting in higher antibiotic resistance.

View full abstract

Studies have shown that linezolid achieves good clinical outcomes against multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis (TB). However, the efficacy of linezolid for individual TB patients and its precise contribution to combination regimens remain unclear for Chinese patients. This study examined the clinical outcomes and safety of linezolid in adults with XDR pulmonary TB at the Chinese PLA 309 hospital. Sixteen XDR-TB patients received linezolid (600 mg daily) in addition to vitamin B6 (50–100 mg daily) as part of their individualized treatment regimens. Of the 16 patients, 14 had received previous treatment for tuberculosis. Sputum samples for all patients showed high colony-forming unit counts when tested by a real-time polymerase chain reaction (PCR). In addition, a high proportion of patients had cavitary lesions in the lungs. Eleven of the 16 patients (68.75%) had successfully completed therapy with documented negative quantitative PCR (qPCR) data and cultures at follow-up (mean = 12 months). Three patients (18.75%) are still receiving treatment, and all 3 have shown clinical and radiographic improvement. Linezolid was discontinued for 2 patients with persistent positive qPCR data and cultures because they developed severe, intractable diarrhea and nausea shortly after beginning treatment. Data indicated that linezolid was a well-tolerated and efficient treatment for XDB-TB in of Chinese patients.

View full abstract