Use of Multiplex Real-Time PCR To Diagnose Scrub Typhus.

Scrub typhus, caused by Orientia tsutsugamushi, is a common cause of acute undifferentiated febrile illness in the Asia-Pacific region. However, its nonspecific clinical manifestation often prevents early diagnosis. We propose the use of PCR and serologic tests as diagnostic tools. Here, we developed a multiplex real-time PCR assay using hydrolysis (TaqMan) probes targeting O. tsutsugamushi 47-kDa, groEL, and human interferon beta (IFN-ß gene) genes to improve early diagnosis of scrub typhus. The amplification efficiency was higher than 94%, and the lower detection limit was 10 copies per reaction. We used a human gene as an internal DNA quality and quantity control. To determine the sensitivity of this PCR assay, we selected patients with confirmed scrub typhus who exhibited a clear 4-fold increase in the level of IgG and/or IgM. The PCR assay result was positive in 45 of 52 patients, indicating a sensitivity of 86.5% (95% confidence interval [CI]: 74.2 to 94.4). The PCR assessment was negative for all 136 non-scrub typhus patients, indicating a specificity of 100% (95% CI: 97.3 to 100). In addition, this test helped diagnose patients with inconclusive immunofluorescence assay (IFA) results and using single blood samples. In conclusion, the real-time PCR assay proposed here is sensitive and specific in diagnosing scrub typhus. Combining PCR and serologic tests will improve the diagnosis of scrub typhus among patients presenting with acute febrile illness.

Scrub Typhus in Northeastern Thailand: Eschar Distribution, Abnormal Electrocardiographic Findings, and Predictors of Fatal Outcome.

Scrub typhus is endemic in Thailand. Of the 495 patients with acute undifferentiated fever studied in Maharat Nakhon Ratchasima Hospital, Nakhon Ratchasima, Thailand, from June 1, 2011, to December 31, 2012, 146 patients (29.5%) had confirmed scrub typhus. The majority of cases were male, farmers, with the mean (±standard deviation) age of 54.1 ± 15.2 years. A total of 59 patients (40.4%) had eschar lesion. The commonest sites for an eschar in male patients were the perineum, inguinal, and buttock area; whereas in females, it was the head and neck area. Abnormal electrocardiogram was found in 39 of 79 patients (49.4%) with sinus tachycardia being the most frequent finding (17, 21.5%). A total of 73 patients (50%) had at least one complication. Myocarditis was the cause of complete heart block in a scrub typhus patient, and he fully recovered after receiving intravenous chloramphenicol treatment. The case fatality rate was 6.2% (nine deaths).The independent predictors for fatal outcome were age over 65 years (odds ratio [OR] = 14.49, 95% confidence interval [CI] = 1.26-166.44, P = 0.03), acute kidney injury (OR = 12.75, 95% CI = 1.77-92.07, P = 0.01), and hyperbilirubinemia (OR = 24.82, 95% CI = 2.12-286.61, P = 0.01). Early diagnosis and prompt appropriate treatment can improve the patient's outcome.

Prevalence and risk factors of acquiring Strongyloides stercoralis infection among patients attending a tertiary hospital in Thailand.

OBJECTIVES: We aimed to determine the prevalence and risk factors for Strongyloides stercoralis infection in adult patients attending Siriraj Hospital, a tertiary hospital in Thailand. METHODS: A case-control study was carried out between July 2008 and April 2010. Case and control were identified from 6022 patients for whom results of faecal examination were available. A case was a patient who had S. stercoralis larva detected from faecal examination. Control was randomly selected from patients without S. stercoralis larvae detected in three consecutive faecal examinations. The proportion of control to case was 2 : 1. Demographic and clinical data for the day of diagnosis and retrospectively up to 15 days preceding the date of faecal examination were reviewed from their medical records. RESULTS: Overall, 149 (2.47%) patients had S. stercoralis larvae positive. There were 105 males (70.5%), with the mean (SD) age of 53.9 (17.2) years. A total of 300 controls were selected. Male gender (odds ratio (OR)  =  2.79, 95% confidence interval (CI) 1.78-4.27)), human immunodeficiency virus (HIV) infection (OR  =  3.23, 95% CI 1.43-7.29), and eosinophilia (OR  =  1.81, 95% CI 1.33-2.47) were found to be independent risk factors associated with S. stercoralis infection in this setting. Corticosteroid or other immunosuppressive treatment, and other concomitant illnesses were not associated with increased risk of S. stercoralis infection. CONCLUSION: In this setting, strongyloidiasis was seen more often in male patients with eosinophilia and with HIV infection. Prevention of fatal complication caused by S. stercoralis by regular faecal examination, or serology for early detection and treatment of undiagnosed S. stercoralis infection, is warranted in these high-risk patients.

Serological response of patients with influenza A (H1N1) pdm09-associated pneumonia: an observational study.

BACKGROUND: Little is known about the dynamics or magnitude of antibody response in patients with influenza A (H1N1) pdm09-associated pneumonia. We described and compared the antibody response to influenza A (H1N1) pdm09 in patients with and without pneumonia. METHODS: We collected serum samples and determined antibody titers by the hemagglutination inhibition (HI) and microneutralization (mNT) assays from patients with RT-PCR confirmed influenza A (H1N1) pdm09 virus at baseline, 1, 2 and 6 months after onset of illness. RESULTS: Fifty-nine patients were enrolled, 45 (76.3%) were between 15 and 60 years of age, 49 (83.1%) were hospitalized and 25 (42.4%) had complications with pneumonia. Ninety-four percent of patients had HI titers ≥ 1: 40 and 90% had mNT titers ≥ 1: 160 at 2 months after illness. Geometric mean titers (GMT) of HI and mNT increased significantly (p<0.001) between baseline and months 1 or 2, then declined significantly (p<0.001) at month 6 by the HI assay, but dropped to an insignificant level (p=0.24) by the mNT assay. The mNT-GMT was at least twice as high as corresponding HI antibodies over a 6 month period. The GMT of HI and mNT in those with pneumonia (1 mo) peaked earlier than that of those without pneumonia (2 mo). When adjusted by age and gender, those with pneumonia had a higher HI-GMT than those without pneumonia at 1 month (264 vs. 117, p=0.007), 2 months (212 vs. 159, p=0.013), and 6 months (160 vs. 82, p=0.018). CONCLUSIONS: The patients recovered from influenza A (H1N1) pdm09-associated pneumonia, clearly developed an earlier and more robust antibody response until 6 months after onset of illness. The results in our study are useful to determine an appropriate donor and timing to obtain convalescent plasma for adjunctive treatment of seriously ill patients with pandemic H1N1 influenza.

Adult-onset immunodeficiency in Thailand and Taiwan.

BACKGROUND: Autoantibodies against interferon-γ are associated with severe disseminated opportunistic infection, but their importance and prevalence are unknown. METHODS: We enrolled 203 persons from sites in Thailand and Taiwan in five groups: 52 patients with disseminated, rapidly or slowly growing, nontuberculous mycobacterial infection (group 1); 45 patients with another opportunistic infection, with or without nontuberculous mycobacterial infection (group 2); 9 patients with disseminated tuberculosis (group 3); 49 patients with pulmonary tuberculosis (group 4); and 48 healthy controls (group 5). Clinical histories were recorded, and blood specimens were obtained. RESULTS: Patients in groups 1 and 2 had CD4+ T-lymphocyte counts that were similar to those in patients in groups 4 and 5, and they were not infected with the human immunodeficiency virus (HIV). Washed cells obtained from patients in groups 1 and 2 had intact cytokine production and a response to cytokine stimulation. In contrast, plasma obtained from these patients inhibited the activity of interferon-γ in normal cells. High-titer anti-interferon-γ autoantibodies were detected in 81% of patients in group 1, 96% of patients in group 2, 11% of patients in group 3, 2% of patients in group 4, and 2% of controls (group 5). Forty other anticytokine autoantibodies were assayed. One patient with cryptococcal meningitis had autoantibodies only against granulocyte-macrophage colony-stimulating factor. No other anticytokine autoantibodies or genetic defects correlated with infections. There was no familial clustering. CONCLUSIONS: Neutralizing anti-interferon-γ autoantibodies were detected in 88% of Asian adults with multiple opportunistic infections and were associated with an adult-onset immunodeficiency akin to that of advanced HIV infection. (Funded by the National Institute of Allergy and Infectious Diseases and the National Institute of Dental and Craniofacial Research; ClinicalTrials.gov number, NCT00814827.).

Performance of SD Bioline Tsutsugamushi assays for the diagnosis of scrub typhus in Thailand.

OBJECTIVE: To assess the diagnostic capacity of a commercially available test (SD Bioline Tsutsugamushi assay) to aid with the diagnosis of scrub typhus in febrile patients in Thailand. MATERIAL AND METHOD: A commercially available lateral-flow-format immunochromatographic test (ICT) for the detection of O. tsutsugamushi IgM, IgG and IgA antibodies was evaluated, using archived serum samples from 102 laboratory confirmed scrub typhus patients and from 63 patients with other causes of fever as the negative control. RESULTS: The sensitivity, specificity of this rapid immunochromatographic test were 66.7% (95% CI, 57.1 to 75.1%) and 98.4% (95% CI 91.5 to 99.7%) respectively. False positive ICT result occurred in one patient with influenza A infection. Among patients with scrub typhus, 17 out of 38 patients (44.7%, 95% CI 30.2 to 60.3%) with negative IgM antibody test by IFA (titer < 1:50) had positive ICT test. Compared to IFA IgG, 33 out of 54 patients (66.1%, 95% CI 47.8 to 72.9%) with negative IgG antibody test by IFA (titer < 1:50) had positive ICT test. CONCLUSION: This rapid ICT test for the diagnosis of scrub typhus was more sensitive than the standard IFA in acute phase specimens.

Performance of Leptospira immunoglobulin M ELISA and rapid immunoglobulin G immunochromatographic assays for the diagnosis of leptospirosis.

Using archived samples, we assessed the diagnostic capacity of two commercially available tests (an ELISA IgM and a rapid immunochromatographic test, ICT) for the detection of Leptospira spp. IgM and IgG antibodies to aid with the diagnosis of acute leptospirosis in febrile patients in Thailand. The sensitivities of the ELISA for the detection of IgM and the rapid immunochromatographic test for the detection of IgG were 60.7% (95% CI, 50.3 to 70.2%), and 83.2% (95% CI 73.9 to 89.6%) respectively. False positive ICT result occurred in one patient with influenza B infection. The positive rates of both assays were high after the first week of onset of fever up to third weeks of illness.

Orientia tsutsugamushi stimulates an original gene expression program in monocytes: relationship with gene expression in patients with scrub typhus.

Orientia tsutsugamushi is the causal agent of scrub typhus, a public health problem in the Asia-Pacific region and a life-threatening disease. O. tsutsugamushi is an obligate intracellular bacterium that mainly infects endothelial cells. We demonstrated here that O. tsutsugamushi also replicated in monocytes isolated from healthy donors. In addition, O. tsutsugamushi altered the expression of more than 4,500 genes, as demonstrated by microarray analysis. The expression of type I interferon, interferon-stimulated genes and genes associated with the M1 polarization of macrophages was significantly upregulated. O. tsutsugamushi also induced the expression of apoptosis-related genes and promoted cell death in a small percentage of monocytes. Live organisms were indispensable to the type I interferon response and apoptosis and enhanced the expression of M1-associated cytokines. These data were related to the transcriptional changes detected in mononuclear cells isolated from patients with scrub typhus. Here, the microarray analyses revealed the upregulation of 613 genes, which included interferon-related genes, and some features of M1 polarization were observed in these patients, similar to what was observed in O. tsutsugamushi-stimulated monocytes in vitro. This is the first report demonstrating that monocytes are clearly polarized in vitro and ex vivo following exposure to O. tsutsugamushi. These results would improve our understanding of the pathogenesis of scrub typhus, during which interferon-mediated activation of monocytes and their subsequent polarization into an M1 phenotype appear critical. This study may give us a clue of new tools for the diagnosis of patients with scrub typhus.

Efficacy and safety of single and double doses of ivermectin versus 7-day high dose albendazole for chronic strongyloidiasis.

BACKGROUND: Strongyloidiasis, caused by an intestinal helminth Strongyloides stercoralis, is common throughout the tropics. It remains an important health problem due to autoinfection, which may result in hyperinfection and disseminated infection in immunosuppressed patients, especially patients receiving chemotherapy or corticosteroid treatment. Ivermectin and albendazole are effective against strongyloidiasis. However, the efficacy and the most effective dosing regimen are to be determined. METHODS: A prospective, randomized, open study was conducted in which a 7-day course of oral albendazole 800 mg daily was compared with a single dose (200 microgram/kilogram body weight), or double doses, given 2 weeks apart, of ivermectin in Thai patients with chronic strongyloidiasis. Patients were followed-up with 2 weeks after initiation of treatment, then 1 month, 3 months, 6 months, 9 months, and 1 year after treatment. Combination of direct microscopic examination of fecal smear, formol-ether concentration method, and modified Koga agar plate culture were used to detect strongyloides larvae in two consecutive fecal samples in each follow-up visit. The primary endpoint was clearance of strongyloides larvae from feces after treatment and at one year follow-up. RESULTS: Ninety patients were included in the analysis (30, 31 and 29 patients in albendazole, single dose, and double doses ivermectin group, respectively). All except one patient in this study had at least one concomitant disease. Diabetes mellitus, systemic lupus erythrematosus, nephrotic syndrome, hematologic malignancy, solid tumor and human immunodeficiency virus infection were common concomitant diseases in these patients. The median (range) duration of follow-up were 19 (2-76) weeks in albendazole group, 39 (2-74) weeks in single dose ivermectin group, and 26 (2-74) weeks in double doses ivermectin group. Parasitological cure rate were 63.3%, 96.8% and 93.1% in albendazole, single dose oral ivermectin, and double doses of oral ivermectin respectively (P = 0.006) in modified intention to treat analysis. No serious adverse event associated with treatment was found in any of the groups. CONCLUSION/SIGNIFICANCE: This study confirms that both a single, and a double dose of oral ivermectin taken two weeks apart, is more effective than a 7-day course of high dose albendazole for patients with chronic infection due to S. stercoralis. Double dose of ivermectin, taken two weeks apart, might be more effective than a single dose in patients with concomitant illness. TRIAL REGISTRATION: ClinicalTrials.gov NCT00765024.