Persistence of dengue genome in a remotely infected patient.

Background: Dengue virus infection is an intriguing illness. It is traditionally thought of as a self-limited and nonpersistent disease. Objectives: We report a case with persistent dengue virus genome detectable in hematopoietic cells of a person with remote infection. Methods: A patient with multiple myeloma in remission was prepared for peripheral blood stem cell (PBSC) transplantation. Plasma and G-CSF-stimulated, mobilized PBSCs were collected. Dengue-specific reverse transcription polymerase chain reaction (RT-PCR) was performed in both pre- and post-stimulated blood specimens. Anti-dengue antibodies by ELISA and by neutralization assay were measured before and after the stem cell mobilization. Results: The viral genome was detected only in the PBSC of the post-G-CSF-stimulated specimens. Anti-dengue antibodies were negative and positive, by ELISA and neutralization assays, respectively, both before and after stem cell mobilization. Conclusion: Our findings reveal a persistent infection. Whether and how this strain may interact with subsequent serotype(s) remains to be elucidated.

Occurrence of mcr-mediated colistin resistance in Salmonella clinical isolates in Thailand.

Nontyphoidal Salmonella, an important zoonotic pathogen and a major cause of foodborne illnesses, could be a potential reservoir of plasmids harbouring mobile colistin resistance gene (mcr). This study reported, for the first time, a high rate of mcr-carrying Salmonella clinical isolates (3.3%, 24/724) in Thailand, associated with mcr-3 gene (3.0%, 22/724) in S. 4,[5],12:i:-(15.4%, 4/26), S. Typhimurium (8.8%, 5/57), and S. Choleraesuis (5.6%, 13/231). Remarkably, the increasing trends of colistin and extended-spectrum cephalosporin resistances have displayed a high agreement over the years, with a dramatic rise in the mcr-carrying Salmonella from 1.1% (6/563) during 2005-2007 to 11.2% (18/161) during 2014-2018 when CTX-M-55 became abundant. Clonal and plasmid analysis revealed that the self-transferable IncA/C and a novel hybrid IncA/C-FIIs MDR plasmids were the major vehicles to disseminate both mcr-3 and blaCTX-M55 genes among diverse Salmonella strains, from as early as 2007. To our knowledge the occurrence of mcr-3 and the co-existence of it with blaCTX-M-55 in S. Choleraesuis are reported here for the first time, leading to clinical concern over the treatment of the invasive salmonellosis. This study provides evidence of the potential reservoirs and vectors in the dissemination of the mcr and highlights the co-selection by colistin and/or cephalosporins.

High prevalence of ceftriaxone resistance among invasive Salmonella enterica serotype Choleraesuis isolates in Thailand: The emergence and increase of CTX-M-55 in ciprofloxacin-resistant S. Choleraesuis isolates.

S. Choleraesuis is a highly invasive zoonotic pathogen that causes a serious systemic infection in humans. The emergence and increase of resistance to ceftriaxone and ciprofloxacin among S. Choleraesuis has become a serious therapeutic problem. The present study demonstrated high frequency of antimicrobial resistance in Salmonella Choleraesuis among 414 nontyphoidal Salmonella isolates from bacteremic patients in Thailand. High rates of ceftriaxone (58.3%) and ciprofloxacin (19.6%) resistances were observed in S. Choleraesuis isolates. The dissemination of the self-transferable blaCTX-M-14-carrying IncFIIs, IncFII, and IncI1 plasmids and blaCMY-2-carrying IncA/C plasmid along with the clonal spread of blaCMY-2-harbouring S. Choleraesuis isolates contributed to the high frequency of resistance to extended-spectrum cephalosporins (ESCs; third- and fourth-generation cephalosporins) during 2005-2007. We reported the first occurrence of ceftazidime-hydrolysing CTX-M-55 in S. Choleraesuis isolates which dramatically increased and became the most abundant CTX-M variant among ESC-resistant S. Choleraesuis isolates during 2012-2016. The spread of clone pulsotype B3 was due to the dissemination of IncA/C plasmids carrying both blaCTX-M-55 and qnrS1 among ciprofloxacin-resistant S. Choleraesuis isolates harbouring D87G in GyrA. These isolates were apparently responsible for the high rates of co-resistance to ESCs and ciprofloxacin (51.3%) during 2012-2016. This study emphasizes the importance to have an action plan to control the dissemination of antimicrobial resistance in S. Choleraesuis since this poses a threat to global health due to travel and trade in animal food products.

Occurrence of a novel class 1 integron harboring qnrVC4 in Salmonella Rissen.

We described qnrVC4 in S. Rissen 166ANSS50, a swine isolate, which was detected in the study on quinolone resistance mechanisms of nontyphoidal Salmonella in Thailand. The isolate was found to harbor a Ì´17-kb non-conjugative plasmid carrying qnrVC4 within 8.91kb of a novel In4-like class 1 integron (In805). It contained the multi-drug resistance gene cassettes of qnrVC4-qacH4-aacA4-cmlA7-blaOXA-10-aadA1-dfrA14 and unusual 3'-CS of mobC-IS6100. This 1014-bp qnrVC4 cassette included with promoter (PqnrVC4: -35 TTGAGA and -10 TAGTCT) showed high homology with qnrVC4 in superintegron of V. cholerae O1 El Tor. The qnrVC4 recombinant plasmid resulted in 4-, 8-, and 16-fold increase in the MICs of nalidixic acid (2-8µg/mL), ciprofloxacin (0.015-0.125µg/mL), and norfloxacin (0.03-0.5µg/mL), respectively. In addition, the backbone plasmid revealed a novel replicon belonging to the MOBQ1 group from the broad-host-range mobilisable IncQ1 plasmid RFS1010 based on relaxase sequences. This is the first known report of qnrVC in Salmonella enterica. The qnrVC4 gene was co-transferred with other resistance genes via a novel plasmid-borne In805. This allowed the spread of this resistance gene to Enterobacteriaceae.

Accuracies of Leuconostoc phenotypic identification: a comparison of API systems and conventional phenotypic assays.

BACKGROUND: Commercial diagnostics are commonly used to identify gram-positive bacteria. Errors have been reported mostly at the species level. We have found certain phenotypic criteria used in API systems which significantly misidentify Leuconostoc, an emerging human pathogen, at the genus level. We also attempt to find practical, conventional phenotypic assays for accurate identification of this group of bacteria. METHODS: Clinical isolates of catalase-negative, gram-positive coccoid or coccobacillary bacteria with non-beta hemolysis in our institute during 1997-2004 were subject to an identification aid by API 20 STREP, following the instruction manual, as an aid to conventional phenotypic tests. Those identified as Leuconostoc by API 20 STREP were re-examined by the same kit and also by API 50 CHL according to the instruction manuals, by our Leuconostoc conventional phenotypic assays, by Leuconostoc- and Lactobacillus-specific PCR's, and, where possible, by 16S rDNA sequence analysis. In addition, catalase-negative gram-positive isolates during 2005-2006 which were resistant to vancomycin at high levels were also evaluated by the same phenotypic and genotypic assays. RESULTS: Out of several thousands of clinical gram-positive isolates, 26 catalase negative gram-positive isolates initially identified as Leuconostoc by API 20 STREP and 7 vancomycin-resistant gram-positive catalase-negative bacteria entered the study. 11 out of the 26 isolates and all the 7 isolates were identified as Leuconostoc by API 20 STREP. Only 5 isolates, however, were confirmed by both genotypic and all defined conventional phenotypic criteria. API 50 CHL also failed to reliably provide accurate identification of Leuconostoc. We have identified key problem tests in API 20 STREP leading to misidentification of the bacteria. A simple, conventional set of phenotypic tests for Leuconostoc identification is proposed. CONCLUSION: The current API systems cannot accurately identify Leuconostoc. Identification of vancomycin-resistant, catalase-negative gram-positive bacteria should be performed by a few practical phenotypic assays, with assistance of genotypic assays where available.

Clinical and epidemiological analyses of human pythiosis in Thailand.

BACKGROUND: Pythiosis is an emerging and life-threatening infectious disease in humans and animals that is caused by the pathogenic oomycete Pythium insidiosum. Human pythiosis is found mostly in Thailand, although disease in animals has been increasingly reported worldwide. Clinical information on human pythiosis is limited, and health care professionals are unfamiliar with the disease, leading to underdiagnosis, delayed treatment, and poor prognosis. METHODS: To retrospectively study the clinical and epidemiological features of human pythiosis, we analyzed clinical data from patients with pythiosis diagnosed during the period of January 1985 through June 2003 at 9 tertiary care hospitals throughout Thailand. RESULTS: A total of 102 cases of human pythiosis were documented nationwide. A substantial proportion (40%) of cases occurred in the last 4 years of the 18-year study interval. Clinical presentations fell into 4 groups: cutaneous/subcutaneous cases (5% of cases), vascular cases (59%), ocular cases (33%), and disseminated cases (3%). Almost all patients with cutaneous/subcutaneous, vascular, and disseminated pythiosis (85%) had underlying thalassemia-hemoglobinopathy syndrome. Most ocular cases (84%) were associated with no underlying disease. A majority of the patients were male (71%), were aged 20-60 years (86%), and reported an agricultural occupation (75%). Regarding treatment outcomes, all patients with disseminated infection died; 78% of patients with vascular disease required limb amputation, and 40% of these patients died; and 79% of patients with ocular pythiosis required enucleation/evisceration. CONCLUSIONS: Here, we report, to our knowledge, the largest case study of human pythiosis. The disease has high rates of morbidity and mortality. Early diagnosis and effective treatment are urgently needed to improve clinical outcomes. Because P. insidiosum is distributed worldwide and can infect healthy individuals, an awareness of human pythiosis should be promoted in Thailand and in other countries.

Establishment of real-time polymerase chain reaction-based assay for quantitation of Epstein-Barr virus DNA in healthy donors and in patients with EBV associated lymphoma.

Epstein-Barr virus (EBV) is associated with several malignancies including nasopharyngeal carcinoma and lymphoma in immunocompromised patients. Quantitative monitoring of EBV DNA in these patients has recently become essential for management of the disease. In the present study the authors developed a rapid and reliable real-time PCR to quantify the EBV DNA in peripheral blood mononuclear cell (PBMC) using hybridization probe technique. The real-time primers and probes in this real-time PCR system were designed based on EBNA-1 sequence. The newly-established real-time PCR demonstrated its high sensitivity (as few as 10 copies of EBV could be detected) and specificity. The intra- and inter-assay variations of the assay were shown to be within a 0.5-log10-difference range. A total of 2 EBV-seronegative, 14 EBV-seropositive healthy donors and 4 patients with PCNSL were enrolled into the study. Our results revealed the median of EBV-DNA in lymphoma patients (7886 copies/10(6) PBMC or 15,150 copies /microg DNA) was higher than that of healthy donors (<10 copies/l0(6) PBMC or <10 copies/microg DNA) with statistic significance (P < 0.01). Assessment of this assay in larger number of donors and patients will provide clinical cut-off values which are essential for monitoring and diagnosis of EBV-associated diseases.

Mycotic aneurysm caused by Burkholderia pseudomallei with negative blood cultures.

We describe a case of bacterial aortitis caused by Burkholderia pseudomallei. This patient presented with prolonged fever and hoarseness of voice. Aneurysm removal with Dacron graft replacement was performed, followed by a prolonged course of antibiotics. The patient has progressed satisfactorily without recurrence of symptoms. Previous case reports are summarized.

Dengue infection: a global concern.

Dengue infection, one of the most important mosquito-borne viral diseases of humans, is now a significant problem in several tropical countries. The disease, caused by the four dengue virus serotypes, ranges from asymptomatic infection, undifferentiated fever, dengue fever (DF) to severe dengue hemorrhagic fever (DHF) with or without shock. DHF is characterized by fever, bleeding diathesis and a tendency to develop a potentially fatal shock syndrome. Hematological findings include vasculopathy, coagulopathy and thrombocytopenia as the most constant findings. During the last twenty-five years, there have been increasing reports of dengue infection with unusual manifestations, mainly with cerebral and hepatic symptoms. Laboratory diagnosis includes virus isolation, serology and detection of dengue ribonucleic acid. Successful treatment, which is mainly supportive, depends on early recognition of the disease and careful monitoring for shock. Prevention depends on control of the mosquito vector. More efforts must be made to understand the pathogenesis of DHF in order to develop a safe and effective dengue vaccine.