The quality and quantity media-cultured mononuclear cell transplantation is safe and effective in ischemic hindlimb mouse model.

Objective: This study was conducted to investigate in vitro proangiogenic and anti-inflammatory phenotypes and functions and the in vivo efficacy and safety of quality and quantity (QQ) media-cultured mononuclear cells (MNCs) compared with standard cultured MNCs from the peripheral blood of patients with chronic limb-threatening ischemia (CLTI) with atherosclerotic risk factors. Methods: Peripheral blood MNCs (PBMNCs) from patients with CLTI were cultured in QQ culture media or standard culture media. Phenotypic analysis of progenitor cells (CD34+CD133+), M2 macrophages (CD206+), and inactivated T regulatory cells (CD4+CD25+CD127+), colony-forming assay, and tube formation assay of QQ media-cultured MNCs (QQMNCs) and PBMNCs, were conducted. Intramuscular transplantation of QQMNCs or PBMNCs was performed in the ischemic hindlimb model. The clinical appearance of ischemic limbs was observed, and blood flow in ischemic limbs was measured using a laser Doppler perfusion imager. Outcomes were compared between the QQMNC and PBMNC groups. Results: Twenty patients with CLTI were included. The mean percentages of CD34+ cells, CD133+ cells, CD34+CD133+ progenitor cells, CD206+ cells, colony-forming cells, and tube formation were significantly higher in the QQMNCs. The mean percentage of CD4+CD25+CD127+ cells was significantly lower in QQMNC. The colony-forming unit count and Dil-acetylated low-density lipoprotein uptake were significantly greater in QQMNCs. The clinical appearance of post-QQMNC-injected limbs was less severe than the appearance of post-PBMNC-injected limbs. Limb perfusion was significantly better in the QQMNCs. Conclusions: Proangiogenic and anti-inflammatory phenotypes of MNCs cultured in QQ culture media were reproducible. Intramuscular QQMNC transplantation was safe and resulted in better reperfusion of ischemic hindlimbs compared with PBMNCs.

Determination of Optimal Diagnostic Cut-Offs for the Naval Medical Research Center Scrub Typhus IgM ELISA in Chiang Rai, Thailand.

In this diagnostic accuracy study, we evaluated data from 135 febrile patients from Chiang Rai, to determine the optimal optical density (OD) cutoffs for an in-house scrub typhus IgM ELISA. Receiver operating characteristic curves were generated using a panel of reference assays, including an IgM immunofluorescence assay (IFA), PCR, in vitro isolation, presence of an eschar, or a combination of these. Altogether, 33 patients (24.4%) were diagnosed as having scrub typhus. Correlation between positivity by IFA and increasing OD values peaked at a cutoff of 2.0, whereas there was little association between positivity by culture or eschar with increasing ELISA cutoffs-cutoffs of 3.0 and 4.0 were demonstrated to be optimal for the total absorbance of the OD at dilutions 1:100, 1:400, 1:1,600, and 1:6,400, for admission and convalescent samples, respectively. The optimal cutoff at a 1:100 dilution was found to be between 1.85 and 2.22 for admission samples and convalescent-phase samples, respectively. Sensitivities for the cutoffs varied from 57.1% to 90.0% depending on the reference test and sample timing, whereas specificities ranged from 85.2% to 99.0%. We therefore recommend a cutoff of around 2.0, depending on the sensitivity and specificity desired in clinical or epidemiological settings. The results demonstrate the ELISA to be a valuable diagnostic tool, suitable for use in resource-limited endemic regions, especially when used in combination with other diagnostic modalities such as the presence of an eschar.

Spotted Fever Rickettsiosis in a Wildlife Researcher in Sabah, Malaysia: A Case Study.

We present evidence for a case of spotted fever rickettsiosis with severe complications in a young adult male. Although spotted fever group rickettsiae (SFGR) have been reported as the most prevalent cause of rickettsiosis in rural areas of Sabah, Malaysia since the 1980s, this is the first detailed case report of suspected SFGR in the state. Current data on the prevalence, type, and thorough clinical reports on complications of SFGR and other rickettsioses in Sabah is lacking and required to raise the awareness of such diseases. There is a need to emphasize the screening of rickettsioses to medical personnel and to encourage the use of appropriate antibiotics as early treatment for nonspecific febrile illnesses in this region. Suspected rickettsioses need to be considered as one of the differential diagnoses for patients presenting with acute febrile illness for laboratory investigations, and early treatment instituted.

Strong interferon-gamma mediated cellular immunity to scrub typhus demonstrated using a novel whole cell antigen ELISpot assay in rhesus macaques and humans.

Scrub typhus is a febrile infection caused by the obligate intracellular bacterium Orientia tsutsugamushi, which causes significant morbidity and mortality across the Asia-Pacific region. The control of this vector-borne disease is challenging due to humans being dead-end hosts, vertical maintenance of the pathogen in the vector itself, and a potentially large rodent reservoir of unclear significance, coupled with a lack of accurate diagnostic tests. Development of an effective vaccine is highly desirable. This however requires better characterization of the natural immune response of this neglected but important disease. Here we implement a novel IFN-γ ELISpot assay as a tool for studying O. tsutsugamushi induced cellular immune responses in an experimental scrub typhus rhesus macaque model and human populations. Whole cell antigen for O. tsutsugamushi (OT-WCA) was prepared by heat inactivation of Karp-strain bacteria. Rhesus macaques were infected intradermally with O. tsutsugamushi. Freshly isolated peripheral blood mononuclear cells (PBMC) from infected (n = 10) and uninfected animals (n = 5) were stimulated with OT-WCA, and IFN-γ secreting cells quantitated by ELISpot assay at five time points over 28 days. PBMC were then assayed from people in a scrub typhus-endemic region of Thailand (n = 105) and responses compared to those from a partially exposed population in a non-endemic region (n = 14), and to a naïve population in UK (n = 12). Mean results at Day 0 prior to O. tsutsugamushi infection were 12 (95% CI 0-25) and 15 (2-27) spot-forming cells (SFC)/106 PBMC for infected and control macaques respectively. Strong O. tsutsugamushi-specific IFN-γ responses were seen post infection, with ELISpot responses 20-fold higher than baseline at Day 7 (mean 235, 95% CI 200-270 SFC/106 PBMC), 105-fold higher at Day 14 (mean 1261, 95% CI 1,097-1,425 SFC/106 PBMC), 125-fold higher at Day 21 (mean 1,498, 95% CI 1,496-1,500 SFC/106 PBMC) and 118-fold higher at Day 28 (mean 1,416, 95% CI 1,306-1,527 SFC/106 PBMC). No significant change was found in the control group at any time point compared to baseline. Humans from a scrub typhus endemic region of Thailand had mean responses of 189 (95% CI 88-290) SFC/106 PBMC compared to mean responses of 40 (95% CI 9-71) SFC/106 PBMC in people from a non-endemic region and 3 (95% CI 0-7) SFC/106 PBMC in naïve controls. In summary, this highly sensitive assay will enable field immunogenicity studies and further characterization of the host response to O. tsutsugamushi, and provides a link between human and animal models to accelerate vaccine development.

Optimal Cutoff and Accuracy of an IgM Enzyme-Linked Immunosorbent Assay for Diagnosis of Acute Scrub Typhus in Northern Thailand: an Alternative Reference Method to the IgM Immunofluorescence Assay.

The enzyme-linked immunosorbent assay (ELISA) has been proposed as an alternative serologic diagnostic test to the indirect immunofluorescence assay (IFA) for scrub typhus. Here, we systematically determine the optimal sample dilution and cutoff optical density (OD) and estimate the accuracy of IgM ELISA using Bayesian latent class models (LCMs). Data from 135 patients with undifferentiated fever were reevaluated using Bayesian LCMs. Every patient was evaluated for the presence of an eschar and tested with a blood culture for Orientia tsutsugamushi, three different PCR assays, and an IgM IFA. The IgM ELISA was performed for every sample at sample dilutions from 1:100 to 1:102,400 using crude whole-cell antigens of the Karp, Kato, and Gilliam strains of O. tsutsugamushi developed by the Naval Medical Research Center. We used Bayesian LCMs to generate unbiased receiver operating characteristic curves and found that the sample dilution of 1:400 was optimal for the IgM ELISA. With the optimal cutoff OD of 1.474 at a sample dilution of 1:400, the IgM ELISA had a sensitivity of 85.7% (95% credible interval [CrI], 77.4% to 86.7%) and a specificity of 98.1% (95% CrI, 97.2% to 100%) using paired samples. For the ELISA, the OD could be determined objectively and quickly, in contrast to the reading of IFA slides, which was both subjective and labor-intensive. The IgM ELISA for scrub typhus has high diagnostic accuracy and is less subjective than the IgM IFA. We suggest that the IgM ELISA may be used as an alternative reference test to the IgM IFA for the serological diagnosis of scrub typhus.

Diagnostic Accuracy Assessment of Immunochromatographic Tests for the Rapid Detection of Antibodies Against Orientia tsutsugamushi Using Paired Acute and Convalescent Specimens.

We assessed the diagnostic accuracy of two immunochromatographic tests (ICTs), the Access Bio CareStart Scrub Typhus test (Somerset, NJ) (IgM), and the SD BIOLINE Tsutsugamushi test (Kyonggi-do, Republic of Korea) (IgG, IgM, or IgA) compared with indirect immunofluorescence assay (IFA) and real-time PCR results as reference tests using 86 paired acute and convalescent specimens from febrile patients. The sensitivity and specificity of the CareStart test were 23.3% (95% confidence interval [CI]: 11.8-38.6) and 81.4% (95% CI: 66.6-91.6), respectively, for acute specimens and 32.6% (95% CI: 19.1-48.5) and 79.1% (95% CI: 64.0-90.0), respectively, for convalescent specimens. For the SD BIOLINE test, sensitivity and specificity were 20.9% (95% CI: 10.0-36.0) and 74.4% (95% CI: 58.8-86.5), respectively, for acute specimens and 76.7% (95% CI: 61.4-88.2) and 76.7% (95% CI: 61.4-88.2), respectively, for convalescent specimens. The poor sensitivity obtained for both ICTs during this study when performed on acute specimens highlights the difficulties in prompt diagnosis of scrub typhus.

Application of ImageJ program to the enumeration of Orientia tsutsugamushi organisms cultured in vitro.

The ImageJ program was applied to the enumeration of Orientia tsutsugamushi organisms in cell culture using indirect immunofluorescence assay (IFA). The highest correlation (r=0.984) was observed between manual counting methods and the ImageJ program (MaxEntropy threshold algorithm). This software-based methodology is cheaper, more standardised and better reproducible than a manual-based approach.