Daniel H. Paris

Scrub Typhus Serologic Testing with the Indirect Immunofluorescence Method as a Diagnostic Gold Standard: A Lack of Consensus Leads to a Lot of Confusion

A review was performed to determine the evidence base for scrub typhus indirect immunofluorescence assay (IFA) methodologies and the criteria for positive results. This review included a total of 109 publications, which comprised 123 eligible studies for analysis (14 publications included 2 substudies). There was considerable underreporting of the IFA methodology and seropositivity criteria used, with most studies using a defined cutoff titer rather than an increase in the titer in paired samples. The choice of positivity cutoff titer varied by country and purpose of the IFA test. This variation limits the comparability of seroprevalence rates between studies and, more seriously, raises questions about the appropriateness of the cutoffs for positive IFA results chosen for diagnosis of acute scrub typhus infection. We suggest that the diagnosis of scrub typhus using IFA should be based on a > or =4-fold increase in the titer in paired serum samples and should only be based on a single sample titer when there is an adequate local evidence base.

Vasoactive effects of Aβ in isolated human cerebrovessels and in a transgenic mouse model of Alzheimer's disease: Role of inflammation

Abstract Aβ peptides are the major protein constituents of Alzheimers disease (AD) senile plaques and also form some deposits in the cerebrovasculature leading to cerebral amyloid angiopathy and hemorrhagic stroke. Functional vascular abnormalities are one of the earlier clinical manifestations in both sporadic and familial forms of AD. Most of the cardiovascular risk factors (for instance, diabetes, hypertension, high cholesterol levels, atherosclerosis and smoking) constitute risk factors for AD as well, suggesting that functional vascular abnormalities may contribute to AD pathology. We studied the effect of Aβ on endothelin-1 induced vasoconstriction in isolated human cerebral arteries collected following rapid autopsies. We report that freshly solubilized Aβ enhances endothelin-1 induced vasoconstriction in isolated human middle cerebral and basilar arteries. The vasoactive effect of Aβ in these large human cerebral arteries is inhibited by NS-398, a selective cyclooxygenase-2 inhibitor and by SB202190, a specific p38 Mitogen Activated Protein Kinase inhibitor suggesting the involvement of a pro-inflammatory pathway. Using a scanner laser Doppler imager, we observed that cerebral blood flow is decreased in the double transgenic APPsw Alzheimer mouse (PS1/APPsw) compared to PS1 littermates and can be improved by chronic treatment with either NS-398 or SB202190. Altogether, our data suggest a link between inflammation and the compromised cerebral hemodynamics in AD.

Unresolved Problems Related to Scrub Typhus: A Seriously Neglected Life-Threatening Disease

Ecology and epidemiology. Scrub typhus is a life-threatening infectious disease that presents as an acute undifferentiated febrile illness. Its agent,Orientia tsutsugamushi, is an obligately intracytosolic bacterium that is transmitted by feeding larval trombiculid mites, which are the reservoir of the agent and the only life stage that feeds on a vertebrate host. Nymphal and adult trombiculid mites live in the soil and feed on the eggs of insects. Mites maintain the organisms by transovarian transmission as well as transtadial transmission through the mite’s lifecycle. Although mites transmit O. tsutsugamushi to vertebrate hosts such as rodents, only a small proportion of uninfected mites acquire Orientia during feeding on infected animals, and the ingested Orientia do not establish disseminated infection in the mites and are not transmitted transovarially to the next generation. It seems that chigger cofeeding on rodents is more relevant for effective mouseto-mite transmission of Orientia than feeding on rickettsemic hosts. Thus, the true role of rodents as reservoirs requires additional investigation, because there may be marked genetic variability in chiggers with respect to the ability to acquire rickettsiae by feeding. Also, rodents may be better considered as dead-end hosts just as humans are rather than a reservoir. However, the evolutionary selection of tremendous antigenic diversity of the immunodominant major 56 kDa surface protein presents an enigma if immune variation plays no role in survival of O. tsutsugamushi. The potential immunomodulatory effects of the saliva of larval mites on the pathogenesis of and immunity to infection with O. tsutsugamushi remain undetermined. One million cases of scrub typhus occur each year with an estimated 10% case fatality rate unless treated appropriately, very likely resulting in more deaths than dengue. Currently, scrub typhus has predominantly been reported from an area extending from the Russian Far East and Korea in the north to northern Australia in the south and Afghanistan in the west, and it includes islands of the western Pacific and Indian Oceans, including Japan, Taiwan, Philippines, Papua New Guinea, Indonesia, and Sri Lanka. This geographical range may be an underrepresentation, because case reports have been published from Africa and South America. The recent isolation of a novel species O. chuto acquired by a patient in Dubai, the detection of another divergentOrientia transmitted to a patient in Chile, and serologic diagnoses of scrub typhus acquired in Africa indicate that a wider geographic distribution and genetic diversity of the genus should be investigated. The burden of disease in rural areas of Asia is large, with studies showing scrub typhus causing up to 20% of febrile hospital admissions, an incidence of infections of greater than 3% of the population monthly, seroprevalence over 50% of the population, despite a significant annual rate of reversion to seronegativity of 50% of cases, and a seroconversion rate of 484 per 1,000 person-years. In 1999, the World Health Organization (WHO) stated, “Scrub typhus is probably one of the most underdiagnosed and underreported febrile illnesses requiring hospitalization in the region.” This opinion remains valid today and could justifiably be adjusted to scrub typhus is probably the single most prevalent, under-recognized, neglected, and severe but easily treatable disease in the world. This ancient disease is currently undergoing increased awareness both because of re-emergence and rising incidence in previously unrecognized areas and improved diagnostic testing. It is apparent that scrub typhus has been recognized to occur frequently now in places where the illness was nearly forgotten, including India, Sri Lanka, the Maldives, and Micronesia. However, scrub typhus has also emerged in regions north of the Yangtse River in China, where it was not known previously. The emergence of scrub typhus should also take into consideration the expansion of farmlands that have generated ideal habitats for trombiculid mites. The potential relationship with global climate change is unclear. Greater recognition in some countries, such as Thailand, Laos, Taiwan, and Japan, may reflect increased medical investigations and application of new diagnostic methods. The critical unresolved issues regarding epidemiology emphasize the need to determine the reasons for emergence and re-emergence and the true incidence of this neglected disease, for which calculated days of disability-adjusted years of life lost (DALYs) have not been determined and to which appropriate attention has not been paid (Table 1). Clinical manifestations. Scrub typhus ranges from a mild to a fatal illness. The early clinical manifestations are an eschar, representing localized cutaneous necrosis at the site of mite feeding (which is not always present), and regional lymphadenopathy followed subsequently by fever, headache, myalgia, generalized lymphadenopathy, cough, gastrointestinal symptoms, transient hearing loss, and rash. Progression of severe scrub typhus may manifest as acute respiratory distress, meningoencephalitis, gastrointestinal bleeding, acute renal failure, hypotensive shock, and coagulopathy. Unresolved clinical issues include the reason for the wide range of occurrence of eschars (7–97%), the reason for rash and severity of illness, and the need for characterization and determination of the mechanisms of the coagulopathy, hemorrhages, interstitial pneumonia, and meningoencephalitis. There is currently no unified approach to assess and stratify *Address correspondence to David H. Walker, Department of Pathology, University of Texas Medical Branch, Galveston, TX 77555-0609. E-mail: dwalker@utmb.edu

Causes of non-malarial fever in Laos: a prospective study

Summary Background Because of reductions in the incidence of Plasmodium falciparum malaria in Laos, identification of the causes of fever in people without malaria, and discussion of the best empirical treatment options, are urgently needed. We aimed to identify the causes of non-malarial acute fever in patients in rural Laos. Methods For this prospective study, we recruited 1938 febrile patients, between May, 2008, and December, 2010, at Luang Namtha provincial hospital in northwest Laos (n=1390), and between September, 2008, and December, 2010, at Salavan provincial hospital in southern Laos (n=548). Eligible participants were aged 5–49 years with fever (≥38°C) lasting 8 days or less and were eligible for malaria testing by national guidelines. Findings With conservative definitions of cause, we assigned 799 (41%) patients a diagnosis. With exclusion of influenza, the top five diagnoses when only one aetiological agent per patient was identified were dengue (156 [8%] of 1927 patients), scrub typhus (122 [7%] of 1871), Japanese encephalitis virus (112 [6%] of 1924), leptospirosis (109 [6%] of 1934), and bacteraemia (43 [2%] of 1938). 115 (32%) of 358 patients at Luang Namtha hospital tested influenza PCR-positive between June and December, 2010, of which influenza B was the most frequently detected strain (n=121 [87%]). Disease frequency differed significantly between the two sites: Japanese encephalitis virus infection (p=0·04), typhoid (p=0·006), and leptospirosis (p=0·001) were more common at Luang Namtha, whereas dengue and malaria were more common at Salavan (all p<0·0001). With use of evidence from southeast Asia when possible, we estimated that azithromycin, doxycycline, ceftriaxone, and ofloxacin would have had significant efficacy for 258 (13%), 240 (12%), 154 (8%), and 41 (2%) of patients, respectively. Interpretation Our findings suggest that a wide range of treatable or preventable pathogens are implicated in non-malarial febrile illness in Laos. Empirical treatment with doxycycline for patients with undifferentiated fever and negative rapid diagnostic tests for malaria and dengue could be an appropriate strategy for rural health workers in Laos. Funding Wellcome Trust, WHO–Western Pacific Region, Foundation for Innovative New Diagnostics, US Centers for Disease Control and Prevention.

Diagnosis of Scrub Typhus

Scrub typhus is transmitted by trombiculid mites and is endemic to East and Southeast Asia and Northern Australia. The clinical syndrome classically consists of a fever, rash, and eschar, but scrub typhus also commonly presents as an undifferentiated fever that requires laboratory confirmation of the diagnosis, usually by indirect fluorescent antibody (IFA) assay. We discuss the limitations of IFA, debate the value of other methods based on antigen detection and nucleic acid amplification, and outline recommendations for future study.

Evaluation of Six Commercial Point-of-Care Tests for Diagnosis of Acute Dengue Infections: the Need for Combining NS1 Antigen and IgM/IgG Antibody Detection To Achieve Acceptable Levels of Accuracy

ABSTRACT Six assays were evaluated in this study to determine their suitability for the diagnosis of acute dengue infection using samples from 259 Sri Lankan patients with acute fevers (99 confirmed dengue cases and 160 patients with other confirmed acute febrile illnesses): (i) the Merlin dengue fever IgG & IgM combo device (Merlin), (ii) the Standard Diagnostics Dengue Duo nonstructural 1 (NS1) antigen and IgG/IgM combo device (Standard Diagnostics, South Korea), (iii) the Biosynex Immunoquick dengue fever IgG and IgM (Biosynex, France) assay, (iv) the Bio-Rad NS1 antigen strip (Bio-Rad, France), (v) the Panbio Dengue Duo IgG/IgM Cassette (Inverness, Australia), and (vi) the Panbio dengue NS1 antigen strip (Inverness, Australia). The median number of days of fever prior to admission sample collection was 5 days (interquartile range, 3 to 7 days). Sensitivity and specificity of the NS1 antigen tests ranged from 49 to 59% and from 93 to 99%, respectively, and sensitivity and sensitivity of the IgM antibody test ranged from 71 to 80% and from 46 to 90%, respectively. Combining the NS1 antigen and IgM antibody results from the Standard Diagnostics Dengue Duo test gave the best compromise of sensitivity and specificity (93% and 89%, respectively) and provided the best sensitivity in patients presenting at different times after fever onset. The Merlin IgM/IgG antibody tests correctly classified 64% and 86% of the primary and secondary dengue infection cases, respectively, and the Standard Diagnostics IgM/IgG antibody tests correctly classified 71% and 83% of the primary and secondary dengue infection cases, respectively. This study provides strong evidence of the value of combining dengue antigen- and antibody-based test results in the rapid diagnostic test (RDT) format for the acute diagnosis of dengue.

Isolation of a Novel Orientia Species ( O. chuto sp. nov.) from a Patient Infected in Dubai

ABSTRACT In July 2006, an Australian tourist returning from Dubai, in the United Arab Emirates (UAE), developed acute scrub typhus. Her signs and symptoms included fever, myalgia, headache, rash, and eschar. Orientia tsutsugamushi serology demonstrated a 4-fold rise in antibody titers in paired serum collections (1:512 to 1:8,192), with the sera reacting strongest against the Gilliam strain antigen. An Orientia species was isolated by the in vitro culture of the patients acute blood taken prior to antibiotic treatment. The gene sequencing of the 16S rRNA gene (rrs), partial 56-kDa gene, and the full open reading frame 47-kDa gene was performed, and comparisons of this new Orientia sp. isolate to previously characterized strains demonstrated significant sequence diversity. The closest homology to the rrs sequence of the new Orientia sp. isolate was with three strains of O. tsutsugamushi (Ikeda, Kato, and Karp), with a nucleotide sequence similarity of 98.5%. The closest homology to the 47-kDa gene sequence was with O. tsutsugamushi strain Gilliam, with a nucleotide similarity of 82.3%, while the closest homology to the 56-kDa gene sequence was with O. tsutsugamushi strain TA686, with a nucleotide similarity of 53.1%. The molecular divergence and geographically unique origin lead us to believe that this organism should be considered a novel species. Therefore, we have proposed the name “Orientia chuto,” and the prototype strain of this species is strain Dubai, named after the location in which the patient was infected.

Incidence and Predictors of Virologic Failure of Antiretroviral Triple-Drug Therapy in a Community-Based Cohort

Highly active antiretroviral therapy fails to reach its recommended goal of sustained suppression of viral replication in a substantial proportion of patients. We analyzed incidence and predictors of virologic failure of the first regimen of a triple-drug combination therapy, including a protease inhibitor and two nucleoside analog reverse transcriptase inhibitors (NRTIs), in 274 HIV-infected patients. Long-term virologic response to combination therapy including salvage regimens was assessed 2.5 years after treatment initiation. During an initial observation period of up to 1.8 years (median, 0.8 years) 152 patients (55%) experienced sustained suppression of HIV-1 RNA to <500 copies/ml. Failure to reduce viral load to <500 copies/ml within 6 months (initial failure) was observed in 51 patients (19%). Independent risk factors for initial failure included higher baseline viral load; addition of a protease inhibitor to an unchanged NRTI regimen; use of saquinavir hard-gel capsules; and longer duration of prior NRTI treatment. Within a median of 7 months viral load rebound above 500 copies/ml occurred in 71 of 223 patients (32%) whose viral load had initially decreased below this threshold. In proportional hazard analysis none of the potential risk factors was significantly associated with viral load rebound. However, in 40 patients (56%) with viral load rebound, incomplete adherence to therapy or treatment interruptions preceded the rebound. Virologic outcome after 2.5 years correlated with initial response to the first regimen: viral load was <500 copies/ml in 88, 55, and 21% of patients with sustained suppression, viral load rebound, and initial failure, respectively.

A Systematic Review of Mortality from Untreated Scrub Typhus (Orientia tsutsugamushi)

Background Scrub typhus, a bacterial infection caused by Orientia tsutsugamushi, is increasingly recognized as an important cause of fever in Asia, with an estimated one million infections occurring each year. Limited access to health care and the disease’s non-specific symptoms mean that many patients are undiagnosed and untreated, but the mortality from untreated scrub typhus is unknown. This review systematically summarizes the literature on the untreated mortality from scrub typhus and disease outcomes. Methodology/Principal Findings A literature search was performed to identify patient series containing untreated patients. Patients were included if they were symptomatic and had a clinical or laboratory diagnosis of scrub typhus and excluded if they were treated with antibiotics. The primary outcome was mortality from untreated scrub typhus and secondary outcomes were total days of fever, clinical symptoms, and laboratory results. A total of 76 studies containing 89 patient series and 19,644 patients were included in the final analysis. The median mortality of all patient series was 6.0% with a wide range (min-max) of 0–70%. Many studies used clinical diagnosis alone and had incomplete data on secondary outcomes. Mortality varied by location and increased with age and in patients with myocarditis, delirium, pneumonitis, or signs of hemorrhage, but not according to sex or the presence of an eschar or meningitis. Duration of fever was shown to be long (median 14.4 days Range (9–19)). Conclusions Results show that the untreated mortality from scrub typhus appears lower than previously reported estimates. More data are required to clarify mortality according to location and host factors, clinical syndromes including myocarditis and central nervous system disease, and in vulnerable mother-child populations. Increased surveillance and improved access to diagnostic tests are required to accurately estimate the untreated mortality of scrub typhus. This information would facilitate reliable quantification of DALYs and guide empirical treatment strategies.

Orientia tsutsugamushi in human scrub typhus eschars shows tropism for dendritic cells and monocytes rather than endothelium.

Scrub typhus is a common and underdiagnosed cause of febrile illness in Southeast Asia, caused by infection with Orientia tsutsugamushi. Inoculation of the organism at a cutaneous mite bite site commonly results in formation of a localized pathological skin reaction termed an eschar. The site of development of the obligate intracellular bacteria within the eschar and the mechanisms of dissemination to cause systemic infection are unclear. Previous postmortem and in vitro reports demonstrated infection of endothelial cells, but recent pathophysiological investigations of typhus patients using surrogate markers of endothelial cell and leucocyte activation indicated a more prevalent host leucocyte than endothelial cell response in vivo. We therefore examined eschar skin biopsies from patients with scrub typhus to determine and characterize the phenotypes of host cells in vivo with intracellular infection by O. tsutsugamushi, using histology, immunohistochemistry, double immunofluorescence confocal laser scanning microscopy and electron microscopy. Immunophenotyping of host leucocytes infected with O. tsutsugamushi showed a tropism for host monocytes and dendritic cells, which were spatially related to different histological zones of the eschar. Infected leucocyte subsets were characterized by expression of HLADR+, with an “inflammatory” monocyte phenotype of CD14/LSP-1/CD68 positive or dendritic cell phenotype of CD1a/DCSIGN/S100/FXIIIa and CD163 positive staining, or occasional CD3 positive T-cells. Endothelial cell infection was rare, and histology did not indicate a widespread inflammatory vasculitis as the cause of the eschar. Infection of dendritic cells and activated inflammatory monocytes offers a potential route for dissemination of O. tsutsugamushi from the initial eschar site. This newly described cellular tropism for O. tsutsugamushi may influence its interaction with local host immune responses.