Pattama Ekpo

Recent developments in laboratory diagnosis of melioidosis.

a Laboratory of Immunology, Chulabhorn Research Institute, Bangkok 10210, Thailand b Department of Microbiology, Faculty of Science, Mahidol Uni6ersity, Rama VI Road, Bangkok 10400, Thailand c Department of Immunology, Faculty of Medicine Siriraj Hospital, Mahidol Uni6ersity, Bangkok, Thailand d Department of Microbiology, Faculty of Medicine, Khon Kaen Uni6ersity, Khon Kaen, Thailand e National Institute of Health, Department of Medical Ser6ices, Ministry of Public Health, Nonthaburi, Thailand f Department of Pathology, Faculty of Medicine Ramathibodi Hospital, Mahidol Uni6ersity, Bangkok, Thailand g Department of Internal Medicine, Faculty of Medicine Siriraj Hospital, Mahidol Uni6ersity, Bangkok, Thailand

Efficacy of cultivated corneal epithelial stem cells for ocular surface reconstruction

Purpose To investigate the clinical outcomes of cultivated corneal limbal epithelial transplantation (CLET) using human amniotic membrane for corneal limbal stem-cell deficiency. Methods Prospective, noncomparative case series. Eighteen patients (19 eyes) with severe ocular surface diseases were chosen to undergo CLET using human amniotic membrane. Twelve eyes received auto-CLET, and seven eyes received allo-CLET. Clinical outcomes of corneal surface epithelialization, conjunctivalization, inflammation, visual acuity, graft status, and complications were observed. Results Corneal epithelium cultivated on amniotic membrane (two to four layers) was positive for molecular markers p63, ABCG2, CK3, and CK12. The mean patient age was 44.7 ± 15.2 years. A successful clinical outcome, defined as corneal epithelialization without central conjunctivalization or severe inflammation, was obtained in 14 (73.7%) of 19 eyes (mean follow-up 26.1 ± 13.5 months; range 6–47). A histopathologic success, defined as absence of goblet cells at the central cornea, was achieved in 12 (63.2%) eyes. Clinical failures occurred in five (26.3%) of 19 eyes, and histopathologic failures occurred in seven (36.8%) of 19 eyes. Survival analysis at 1 year showed that the clinical success rate was 77.9% and the pathological success rate was 72.3%. Fourteen of 19 (73.7%) eyes had visual acuity improvements after CLET. Six cases underwent penetrating keratoplasty; five of these grafts remained clear after 20.4 ± 6.9 months (range, 12–31) of follow-up. Complications included infectious keratitis (three cases) and recurrent symblepharon (one case). All complicated cases had lid abnormalities. Factors affecting the final clinical outcomes were lid abnormalities, abnormal corneal stromal beds, and complications. Conclusion CLET can successfully restore ocular surface damage in most cases with corneal limbal stem cell deficiency.

Expression of TNF-α, TGF-β, IP-10 and IL-10 mRNA in kidneys of hamsters infected with pathogenic Leptospira

Leptospirosis is a worldwide zoonosis caused by pathogenic Leptospira. Although several components of this organism have been identified, the molecular mechanisms underlying pathogenesis of this infectious disease are still poorly understood. Besides, direct injury by microbial factors, cytokines produced in response to infection have been proposed to be involved in pathogenesis of leptospirosis. In this study, cytokine gene expression in kidneys was investigated. Hamsters were injected with pathogenic Leptospira interrogans serovar Pyrogenes and were sacrificed on days 3, 5 and 7 after infection. RNA was extracted from kidney tissues. Real-time PCR was performed to demonstrate expression of TNF-alpha, TGF-beta, IP-10 and IL-10 mRNA in kidneys. TNF-alpha, TGF-beta and IP-10 expression could be demonstrated since day 3 post-infection whereas IL-10 expression was detected later on day 5. Leptospira infection resulted in not only expression of a proinflammatory cytokine, TNF-alpha, but also a T cell chemokine, IP-10. Detection of IP-10 suggested the involvement of T cell recruitment in the immune response or pathology in infected kidneys. Expressions of anti-inflammatory cytokines, TGF-beta and IL-10 were also observed. However, the level of TGF-beta expression was prominent since day 3 post-infection whereas IL-10 expression was clearly observed on day 5. Further experiments will provide additional information whether there is a correlation between the expression of these cytokines and pathologies found in an affected organ.

Development of New, Broadly Reactive, Rapid IgG and IgM Lateral Flow Assays for Diagnosis of Scrub Typhus

We evaluated the diagnostic accuracy of two broadly reactive rapid immunochromatographic tests (ICTs) for detection of IgM and IgG against Orientia tsutsugamushi by using archived acute-phase serum samples from 102 patients with laboratory-confirmed scrub typhus, and from 62 archived serum samples from patients with other causes of fever as a negative control. These ICTs were constructed by using a mixture of recombinant proteins: 1) C1, a chimeric protein containing epitopes of the 56-kD antigen from Karp and TA763 strains; 2) Ktr56; and 3) Gmr56. Sensitivities of the ICTs for detection of IgM and IgG were 90.2% (95% confidence interval [CI] = 84.4-96.0%) and 86.3% (95% CI = 80.9-93.8%), respectively. Specificities were 85.5% (95% CI = 73.9-92.2%) and 96.8% (95% CI = 90.3-100%), respectively. Both assays were more sensitive and specific than the standard immune immunofluorescence assay for the early diagnosis of scrub typhus.

Use of Protein-Specific Monoclonal Antibody-Based Latex Agglutination for Rapid Diagnosis of Burkholderia pseudomallei Infection in Patients with Community-Acquired Septicemia

ABSTRACT A latex agglutination test employing monoclonal antibody specific to a 30-kDa protein of Burkholderia pseudomallei was used to detect the organisms in blood culture specimens from 1,139 patients with community-acquired septicemia. The sensitivity, specificity, and positive and negative predictive values of the test were 96.75%, 99.61%, 96.75%, and 99.61%, respectively.

Broad-coverage molecular epidemiology of Orientia tsutsugamushi in Thailand.

Orientia tsutsugamushi, an obligate intracellular bacterium closely related to the genus Rickettsia, is the causative agent of scrub typhus, a major cause of febrile illness in rural areas of Asia-Pacific region. Scrub typhus is transmitted by the bite of infected mites of the genus Leptotrombidium. The region of the 56-kDa TSA gene spanning from variable domain I (VDI) to variable domain IV (VDIV) was sequenced and used for genotyping 77 O. tsutsugamushi samples from human patients confirmed with scrub typhus from 2001 to 2003 and 2009 to 2010 in different regions of Thailand. These sequences were also compared to previously published 56-kDa TSA sequences. Only 4 genotypes out of 8 previously reported in Thailand were identified, i.e. Karp, JG-v, TA763 and Kato, respectively. Two strains were not associated with known genotypes but were closely related to Taiwanese strains. The Karp genotype was confirmed as the predominant clade. The JG-v and TA763 genotypes, in contrast to other studies, also were found. The genotype TA716 was not found, except for one strain previously described.

Orientia tsutsugamushi, agent of scrub typhus, displays a single metapopulation with maintenance of ancestral haplotypes throughout continental South East Asia

Orientia tsutsugamushi is the causative agent of scrub typhus, a major cause of febrile illness in rural area of Asia-Pacific region. A multi-locus sequence typing (MLST) analysis was performed on strains isolated from human patients from 3 countries in Southeast Asia: Cambodia, Vietnam and Thailand. The phylogeny of the 56-kDa protein encoding gene was analyzed on the same strains and showed a structured topology with genetically distinct clusters. MLST analysis did not lead to the same conclusion. DNA polymorphism and phylogeny of individual gene loci indicated a significant level of recombination and genetic diversity whereas the ST distribution indicated the presence of isolated patches. No correlation was found with the geographic origin. This work suggests that weak divergence in core genome and ancestral haplotypes are maintained by permanent recombination in mites while the 56-kDa protein gene is diverging in higher speed due to selection by the mammalian immune system.

Mimotope identification from monoclonal antibodies of Burkholderia pseudomallei using random peptide phage libraries.

This study used random peptide libraries, displayed by bacteriophage T7 and M13, to identify mimotopes from four monoclonal antibodies (mAbs) specific to Burkholderia pseudomallei. Bound phages, selected from fourth-round panning with each mAb, were tested for binding specificity with each mAb using ELISA, before being further amplified and checked for phage peptide sequence using PCR and DNA sequencing. Overall, 75 of 90 phages (83.3%) were ELISA-positive with each mAb. Mimotopes from all 75 phages (100%) were found to match protein sequences of Burkholderia spp. from GenBank. The predominant mimotopes were TP-GRTRVT found in 13.3%, LTPCGRTxD (8%), AREVTLL (6.7%), NxVxKVVSR (5.3%), PCAPRSS (4%), LGRVLAN (4%), RNPKKA (2.7%) and CPYPR (2.7%). The following GenBank-matched proteins (i.e. the hypothetical proteins) were located at the outer membrane of Burkholderia spp.: BPSL2046 of B. pseudomallei K96243 (matched with mimotope CGRTxD), BpseP_02000035 (matched with LGRVLAN), BPSS0784 of B. pseudomallei K96243 (matched with CPYPR), BURPS1710b_1104 of B. pseudomallei 1710b (matched with CARQY) and TonB-dependent siderophore receptor of B. cenocepacia H12424 (matched with CVRxxLTPC and TPCRxRT). These phage mimotopes and matched proteins may have the potential for further use as diagnostic reagent and immunogen against melioidosis. These results demonstrate that phage-display technique has the potential for rapidly identifying phage mimotopes that interact with B. pseudomallei mAbs.

Suspected Mycoplasma Contamination in the Study “Toll-Like Receptor 2 Recognizes Orientia tsutsugamushi and Increases Susceptibility to Murine Experimental Scrub Typhus”

In a recent publication ([1][1]), Gharaibeh et al. demonstrated that human Toll-like receptor 2 (TLR2) recognizes Orientia tsutsugamushi by using TLR2-transfected HEK293 cells. They demonstrated that murine TLR2 is required for inflammatory cytokine production by bone marrow-derived dendritic cells