Role of Burkholderia pseudomallei–Specific IgG2 in Adults with Acute Melioidosis,Thailand

Melioidosis is a life-threatening infectious disease caused by the gram-negative bacillus Burkholderia pseudomallei. An effective vaccine is needed, but data on protective immune responses in human melioidosis are lacking. We used ELISA and an antibody-dependent cellular phagocytosis assay to identify the major features of protective antibodies in patients with acute melioidosis in Thailand. We found that high levels of B. pseudomallei–specific IgG2 are associated with protection against death in a multivariable logistic regression analysis adjusting for age, diabetes, renal disease, and neutrophil count. Serum from melioidosis survivors enhanced bacteria uptake into human monocytes expressing FcγRIIa-H/R131, an intermediate-affinity IgG2-receptor, compared with serum from nonsurvivors. We did not find this enhancement when using monocytes carrying the low IgG2–affinity FcγRIIa-R131 allele. The findings indicate the importance of IgG2 in protection against death in human melioidosis, a crucial finding for antibody-based therapeutics and vaccine development.     

Differential patterns of endothelial and leucocyte activation in 'typhus-like' illnesses in Laos and Thailand

Scrub typhus is responsible for a large proportion of undifferentiated fevers in south-east Asia. The cellular tropism and pathophysiology of the causative agent, Orientia tsutsugamushi, remain poorly understood. We measured endothelial and leucocyte activation by soluble cell adhesion molecule enzyme-linked immunosorbent assays in 242 Lao and Thai patients with scrub or murine typhus, leptospirosis, dengue, typhoid and uncomplicated falciparum malaria on admission to hospital. Soluble E-selectin (sE-selectin) levels were lowest in dengue, sL-selectin highest in scrub typhus with a high sE-selectin to sL-selectin ratio in leptospirosis patients. In scrub typhus patients elevated sL-selectin levels correlated with the duration of skin rash (P = 0.03) and the presence of eschar (P = 0.03), elevated white blood cell (WBC) count (P = 0.007), elevated lymphocyte (P = 0.007) and neutrophil counts (P = 0.015) and elevated levels of sE-selectin correlated with the duration of illness before admission (P = 0.03), the presence of lymphadenopathy (P = 0.033) and eschar (P = 0.03), elevated WBC (P = 0.005) and neutrophil counts (P = 0.0003). In comparison, soluble selectin levels in murine typhus patients correlated only with elevated WBC counts (P = 0.03 for sE-selectin and sL-selectin). Soluble intercellular adhesion molecule-1 and soluble vascular adhesion molecule-1 levels were not associated significantly with any clinical parameters in scrub or murine typhus patients. The data presented suggest mononuclear cell activation in scrub typhus. As adhesion molecules direct leucocyte migration and induce inflammatory and immune responses, this may represent O. tsutsugamushi tropism during early dissemination, or local immune activation within the eschar.     

Evaluation of the Panbio dengue virus nonstructural 1 antigen detection and immunoglobulin M antibody enzyme-linked immunosorbent assays for the diagnosis of acute dengue infections in Laos

We evaluated 2 commercial enzyme-linked immunosorbent assays (ELISAs) for the diagnosis of dengue infection; one a serologic test for immunoglobulin M (IgM) antibodies, the other based on detection of dengue virus nonstructural 1 (NS1) antigen. Using gold standard reference serology on paired sera, 41% (38/92 patients) were dengue confirmed, with 4 (11%) acute primary and 33 (87%) acute secondary infections (1 was of indeterminate status). Sensitivity of the NS1-ELISA was 63% (95% confidence interval [CI], 53-73) on admission samples but was much less sensitive (5%; 95% CI, 1-10) on convalescent samples. The IgM capture ELISA had a lower but statistically equivalent sensitivity compared with the NS1-ELISA for admission samples (45%; 95% CI, 35-55) but was more sensitive on convalescent samples (58%; 95% CI, 48-68). The results of the NS1 and IgM capture ELISAs were combined using a logical OR operator, increasing the sensitivity for admission samples (79%; 95% CI, 71-87), convalescent samples (63%; 95% CI, 53-73), and all samples (71%; 95% CI, 65-78).     

Accuracy of AccessBio Immunoglobulin M and Total Antibody Rapid Immunochromatographic Assays for the Diagnosis of Acute Scrub Typhus Infection

Using archived samples, we assessed the diagnostic capacity of a rapid immunochromatographic test (ICT) for the detection of Orientia tsutsugamushi IgM and total antibodies to aid with the diagnosis of acute scrub typhus infection in febrile patients in Laos. The sensitivity and the specificity of the ICT for the detection of IgM were 96.8% (121/125 samples; 95% confidence interval [CI], 92.1 to 99.1%) and 93.3% (98/105 samples; 95% CI, 86.7 to 97.3%), respectively. For the detection of total antibodies, the sensitivity was 97.6% (122/125 samples; 95% CI, 93.1 to 99.5%), but the specificity was much lower, at 71.4% (75/105 samples; 95% CI, 61.8 to 79.8%).Scrub typhus, caused by Orientia tsutsugamushi, is an important acute febrile illness in the Asia-Pacific region. As very few health facilities have accessible accurate diagnostic tests, the diagnosis of scrub fever must be based on clinical features. However, this is difficult because the clinical symptoms and signs are similar to those of many other febrile diseases, such as murine typhus, leptospirosis, and dengue virus infection. The diagnosis of scrub typhus infection has relied on the detection of O. tsutsugamushi antibodies during the acute phase of the disease, and the “gold standard” assay is the indirect immunofluorescence antibody assay (IFA) (9). The development of rapid, diagnostic tests by the use of immunochromatographic test (ICT) technologies has provided a mechanism for point-of-care serological testing. The objective of the study described here was to assess the diagnostic capacities of two commercial rapid ICTs for the detection of O. tsutsugamushi IgM and whole antibodies to aid with the diagnosis of acute scrub typhus infection by the use of stored, characterized sera collected from febrile patients in the tropical environment of the Lao People''s Democratic Republic (Laos) and Thailand where scrub typhus is endemic.     

Serological and blood culture investigations of Nepalese fever patients

Serological testing of paired (i.e. admission and convalescent) sera from 103 fever patients in Kathmandu, Nepal, was performed to estimate the prevalence rates of scrub typhus, murine typhus, Leptospira and dengue virus antibodies and to determine their role in the cause of active infections. Blood cultures from 15 patients grew Salmonella enterica serovar Typhi, 8 grew S. Paratyphi A and 6 grew other bacteria. Diagnostic antibody levels were detected against murine typhus (27/103; 26%), scrub typhus (23/103; 22%), Leptospira (10/103; 10%) and dengue virus (8/103; 8%). Nineteen patients (18%) had diagnostically raised antibodies to more than one infectious agent. Seven S. Typhi (7/15; 47%) and two S. Paratyphi A (2/8; 25%) patients had significant scrub typhus, murine typhus, Leptospira or dengue virus IgM antibody titres. This study confirms the presence of leptospiral, rickettsial and dengue infections in Kathmandu as well as evidence for mixed infections with S. Typhi and Orientia tsutsugamushi or Rickettsia typhi. These infections should be kept in mind when considering the differential diagnoses of fever and empirical treatment options in Nepal. Many patients demonstrated static IgM antibody results between paired serum collections, suggesting recent rather than acutely active infections.     

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

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.     

Accuracy of Rapid IgM-Based Immunochromatographic and Immunoblot Assays for Diagnosis of Acute Scrub Typhus and Murine Typhus Infections in Laos

We studied the diagnostic accuracy of a rapid immunochromatographic test (ICT) for detection of IgM against scrub typhus (ST ICT) and an immunoblot test for the detection of IgM against murine typhus (MT IBT) by using admission serum samples from 1,030 febrile patients in Laos. Sensitivity and specificity for the ST ICT determined by using the diagnostic criteria of a four-fold increase in IgM against Orientia tsutsugamushi between paired samples were 23.8% (95% confidence interval [CI] = 15.9–33.3%) and 86.2% (95% CI = 84.1–88.6%), respectively. Sensitivity and specificity for the ST ICT determined by using an admission IgM titer ≥ 1:400 were 39.1% (95% CI = 34.1–44.2%) and 99.5% (95% CI = 98.7–99.9%), respectively. Sensitivity and specificity for the MT IBT determined by using the criteria of a four-fold increase in IgM against Rickettsia typhi between paired serum samples were 61.2% (95% CI = 53.7–68.3%) and 86.5% (95% CI = 84.1–88.8%), respectively. Sensitivity and specificity for the MT IBT determined by using an admission IgM titer ≥ 1:400 were 54.6% (95% CI = 49.1–60.0%) and 94.1% (95% CI = 92.0–95.7%), respectively. Both assays had relatively good specificity but low sensitivity and thus have limited utility for admission diagnosis.     

Diabetes alters immune response patterns to acute melioidosis in humans

Diabetes mellitus (DM) is a serious global health problem currently affecting over 450 million people worldwide. Defining its interaction with major global infections is an international public health priority. Melioidosis is caused by Burkholderia pseudomallei, an exemplar pathogen for studying intracellular bacterial infection in the context of DM due to the 12‐fold increased risk in this group. We characterized immune correlates of survival in peripheral blood of acute melioidosis patients with and without DM and highlight different immune response patterns. We demonstrate the importance of circulating NK cells and show that CX3CR1 expression on lymphocytes is a novel correlate of survival from acute melioidosis. Furthermore, excessive serum levels of IL‐15 and IL‐18BP contribute to poor outcome independent of DM comorbidity. CD8⁺ T cells and granzyme B expression in NK cells are important for survival of non‐DM patients, whereas high antibody titers against B. pseudomallei and double‐negative T cells are linked to survival of DM patients. Recall responses support a role of γδ T‐cell‐derived IFN‐γ in the establishment of protective immunity in the DM group. Defining the hallmarks of protection in people with DM is crucial for the design of new therapies and vaccines targeting this rapidly expanding risk group.     

A highly sensitive quantitative real-time PCR assay based on the groEL gene of contemporary Thai strains of Orientia tsutsugamushi

Partial nucleotide sequences (459 bp) of the groEL gene (encoding the 60-kDa heat shock protein, HSP60) from 23 contemporary isolates of Orientia tsutsugamushi isolated from patients with acute scrub typhus in Thailand were compared with 16 reference strain sequences to evaluate the potential of groEL as a conserved and representative target for molecular diagnostics.. Overall nucleotide identity within all available O. tsutsugamushi isolates ( n  = 39) was 98.8% (range: 95.0–100), reflecting a high degree of conservation; nucleotide identities were 67.5% and 65.6%, respectively, when typhus and spotted fever group rickettsiae were included.. A highly sensitive and quantitative real-time PCR assay was designed and evaluated using 61 samples, including buffy coats from patients in Thailand and Laos. Reliable and accurate quantitation of bacterial loads allows further investigation of other diagnostic methods and may lead to an improved understanding of the pathophysiology of acute scrub typhus, an important but under-recognized disease.