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.

An ELISA assay using a combination of recombinant proteins from multiple strains of Orientia tsutsugamushi offers an accurate diagnosis for scrub typhus.

BACKGROUND: Scrub typhus (ST) is a disease caused by an obligate intracellular bacterium, Orientia tsutsugamushi, an organism that requires a BSL3 laboratory for propagation. The disease is hallmarked by an eschar at the site of the chigger bite, followed by the development of fever, malaise, myalgia, anorexia, and papulomacular rash. Indirect immunofluorescent assay (IFA) is the gold standard for scrub typhus diagnosis, however, the subjectivity of the assay, the need for a specialized laboratory and instruments has limited the wide use of the test in resource limited areas. METHODS: A recombinant-protein based enzyme linked immunosorbent assay (ELISA) using the most abundant and immunodominant protein for the detection of Orientia specific antibodies in serum has been developed. The performance of the assay was evaluated using prospectively collected acute sera from 248 randomly selected patients in Thailand. The ELISA assay was evaluated using two different cutoff values. RESULTS: The receiver operating characteristic (ROC) curve generated cutoff values gave slightly better consistency with diagnosis of ST than those cutoff values established by averaging ELISA optical density of known negatives at 99% confidence interval. Both cutoff values provided similar statistical parameters when compared with the diagnosis of ST, indicating the validity of both calculations to derive cutoff values. These results suggest that both IgG and IgM ELISA performed well to accurately diagnose scrub typhus cases in endemic areas using only acute serum samples. CONCLUSIONS: We have successfully developed an ELISA assay for the detection of Orientia-specific antibodies in serum that could provide effective screening of acute sera under clinical setup and it is also a useful assay to estimate seroprevalence in various endemic areas.

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.

Performance of a New Microfluidic Dengue NS1 Immuno-magnetic Agglutination Assay for the Rapid Diagnosis of Dengue Infection in Adults.

Dengue (DENV) infections are a public health concern worldwide and thus early diagnosis is important to ensure appropriate clinical management. The rapid diagnostic test (RDT) targets nonstructural protein 1 (NS1) detection and is the main tool used for diagnostic purpose. In this study, we evaluated the performance of a new rapid and semi-quantitative microfluidic DENV NS1 immuno-magnetic agglutination assay or IMA (ViroTrack Dengue Acute, BluSense Diagnostics, Copenhagen, Denmark). We studied 233 subjects confirmed to have DENV infection (by a real-time reverse transcriptase polymerase chain reaction) and 200 control samples were taken from patients with confirmed diagnoses of other febrile illnesses, in Thailand. Samples were tested using the NS1 antigen (Ag) detection methods: in-house NS1 Ag ELISA (ELISA), SD BIOLINE Dengue NS1 Ag RDT (ICT), and ViroTrack Dengue Acute (IMA). Sensitivities of these tests were 86.3%, 78.9%, and 85.5%, respectively. All tests showed high specificity (100%, 99%, and 97% for ELISA, ICT, and IMA, respectively). The sensitivities of both RDTs were affected by the low sensitivity to DENV-2 and DENV-4. NS1 Ag was detected in every patient on day 1 and day 2 after onset of illness by ELISA and IMA with a decline in detection rates over time after day 6 of illness. NS1 detection rate using ICT decreased from 100% on day 1 of illness to 98.6% on day 2 after onset of illness. By day 6, the detection rate was 45.9%. Thus, IMA performed better than ICT for early and rapid diagnosis of DENV infections in endemic countries.

Efficacy and safety of a single-dose veterinary preparation of ivermectin versus 7-day high-dose albendazole for chronic strongyloidiasis.

A prospective, randomised, open study compared a 7-day course of oral albendazole 800 mg daily with a single oral dose of the parenteral veterinary preparation of ivermectin in 42 Thai patients with chronic strongyloidiasis (21 in each group). The primary endpoints were relief of symptoms (if present) and clearance of Strongyloides larvae from faeces immediately after treatment and at follow-up to 16 weeks later. Cure rates in the albendazole and ivermectin groups were 38.1% and 76.2%, respectively, (P=0.029) in the intention-to-treat analysis and 50% and 88.9%, respectively, (P=0.023) in the per-protocol analysis. Acute generalised exanthematous pustulosis developed in one patient who was treated with the veterinary preparation of ivermectin. This study confirms the superiority of ivermectin compared with albendazole as well as that oral use of the parenteral veterinary preparation in humans is as effective and safe as human preparations.

Radiographic chest findings and clinical correlations in leptospirosis.

OBJECTIVE: To determine the clinical presentations, radiographic chest findings, and their correlation in patients with leptospirosis. DESIGN: A cross sectional study. SETTING: Between July 2001- December 2002 at 3 hospitals in North Eastern Thailand. MATERIAL AND METHOD: Two hundred and forty patients with laboratory confirmed leptospirosis. RESULTS: Two hundred and nine (87.1%) patients were males. The mean age was 37.53 years (range 13-76). The median duration of fever was 3 days (range 1-13). Overall, 154 patients (64.2%) had respiratory symptoms and 26 (10.8%) patients had hemoptysis. Jaundice was detected in 76 (31.7%) patients, hypotension in 50 (20.8%), renal dysfunction in 80 (30%), and multiorgan dysfunction in 62 (25.8%) on admission. One hundred and fifty-four (64.17%) patients had abnormal chest radiographs on admission (classified as cardiovascular, pulmonary, and mixed cardio-pulmonary involvement in 40 (25.97%), 41 (26.62%), and 73 (47.4%) patients, respectively). Jaundice was significantly associated with the likelihood of having abnormal chest radiography on admission. Air- space nodules detected on the chest radiograph were significantly more common in patients with renal dysfunction and patients who required mechanical ventilation. CONCLUSION: Pulmonary and cardiovascular involvements are common in leptospirosis. Air-space nodules detected by chest radiography may indicate severe leptospirosis.

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.

Human leptospirosis trends: northeast Thailand, 2001-2012.

The objective of this study was to determine the changing trend of leptospirosis over time in Thailand using two prospective hospital-based studies conducted amongst adult patients with acute undifferentiated fever (AUFI) admitted to Maharat Nakhon Ratchasima Hospital, Nakhon Ratchasima Province, Thailand between July 2001 to December 2002 and between July 2011 to December 2012. During the first period, leptospirosis (98 patients, 40%) and scrub typhus (59 patients, 24.1%) were the two major causes of AUFI. In the second period, scrub typhus (137 patients, 28.3%) was found to be more common than leptospirosis (61 patients, 12.7%). Amongst patients with leptospirosis, the proportion of male patients and the median age were similar. Leptospira interrogans serogroup Autumnalis was the major infecting serogroup in both study periods. The case fatality rate of leptospirosis was significantly higher in 2011-2012 as compared with the case fatality rate in 2001-2002 (19.7% vs. 6.3%, p < 0.001). In summary, we found that number of leptospirosis cases had decreased over time. This trend is similar to reportable data for leptospirosis complied from passive surveillance by the Ministry of Public Health, Thailand. However, the case fatality rate of severe leptospirosis has increased. Severe lung hemorrhage associated with leptospirosis remained the major cause of death.

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.

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.

Strategies for diagnosis and treatment of suspected leptospirosis: a cost-benefit analysis.

BACKGROUND: Symptoms and signs of leptospirosis are non-specific. Several diagnostic tests for leptospirosis are available and in some instances are being used prior to treatment of leptospirosis-suspected patients. There is therefore a need to evaluate the cost-effectiveness of the different treatment strategies in order to avoid misuse of scarce resources and ensure best possible health outcomes for patients. METHODS: The study population was adult patients, presented with uncomplicated acute febrile illness, without an obvious focus of infection or malaria or typical dengue infection. We compared the cost and effectiveness of 5 management strategies: 1) no patients tested or given antibiotic treatment; 2) all patients given empirical doxycycline treatment; patients given doxycycline when a patient is tested positive for leptospirosis using: 3) lateral flow; 4) MCAT; 5) latex test. The framework used is a cost-benefit analysis, accounting for all direct medical costs in diagnosing and treating patients suspected of leptospirosis. Outcomes are measured in length of fever after treatment which is then converted to productivity losses to capture the full economic costs. FINDINGS: Empirical doxycycline treatment was the most efficient strategy, being both the least costly alternative and the one that resulted in the shortest duration of fever. The limited sensitivity of all three diagnostic tests implied that their use to guide treatment was not cost-effective. The most influential parameter driving these results was the cost of treating patients with complications for patients who did not receive adequate treatment as a result of incorrect diagnosis or a strategy of no-antibiotic-treatment. CONCLUSIONS: Clinicians should continue treating suspected cases of leptospirosis on an empirical basis. This conclusion holds true as long as policy makers are not prioritizing the reduction of use of antibiotics, in which case the use of the latex test would be the most efficient strategy.

Doxycycline versus azithromycin for treatment of leptospirosis and scrub typhus.

Leptospirosis and scrub typhus are important causes of acute fever in Southeast Asia. Options for empirical therapy include doxycycline and azithromycin, but it is unclear whether their efficacies are equivalent. We conducted a multicenter, open, randomized controlled trial with adult patients presenting with acute fever (<15 days), without an obvious focus of infection, at four hospitals in Thailand between July 2003 and January 2005. Patients were randomly allocated to receive either a 7-day course of doxycycline or a 3-day course of azithromycin. The cure rate, fever clearance time, and adverse drug events were compared between the two study groups. A total of 296 patients were enrolled in the study. The cause of acute fever was determined for 151 patients (51%): 69 patients (23.3%) had leptospirosis; 57 patients (19.3%) had scrub typhus; 14 patients (4.7%) had murine typhus; and 11 patients (3.7%) had evidence of both leptospirosis and a rickettsial infection. The efficacy of azithromycin was not inferior to that of doxycycline for the treatment of both leptospirosis and scrub typhus, with comparable fever clearance times in the two treatment arms. Adverse events occurred more frequently in the doxycycline group than in the azithromycin group (27.6% and 10.6%, respectively; P = 0.02). In conclusion, doxycycline is an affordable and effective choice for the treatment of both leptospirosis and scrub typhus. Azithromycin was better tolerated than doxycycline but is more expensive and less readily available.