Efficacy of Bortezomib for Treating Anti-Interferon-Gamma Autoantibody-Associated Adult-Onset Immunodeficiency Syndrome.

BACKGROUND: Currently, there is no effective treatment for adult-onset immunodeficiency (AOID) syndrome with anti-interferon-gamma autoantibodies (anti-IFN-γ-auto-Abs). This study aimed to investigate the effectiveness of bortezomib (BTZ) for decreasing anti-IFN-γ-auto-Abs. METHODS: A pre- and post-intervention study was conducted from February 2017 through June 2019 at Siriraj Hospital (Bangkok, Thailand). Five patients were invited to receive once-weekly BTZ (1.3 mg/m2 body surface area) subcutaneously for 8 weeks followed by oral cyclophosphamide (1 mg/kg/d) for 4 months. The primary outcomes were the difference in antibody level at 8 and 48 weeks compared with baseline and the incidence of serious adverse events (AEs). The secondary outcome was the occurrence of opportunistic infections (OIs) during the 72 weeks after starting BTZ. RESULTS: The median patient age was 46 years (range, 34-53). All patients had 3-5 OIs prior to enrollment. All patients were receiving antimycobacterial agents for treatment of nontuberculous mycobacterial infection at enrollment. There was no significant difference in the mean optical density of auto-Abs at 8 weeks (3.73 ± 0.72) or 48 weeks (3.74 ± 0.53) compared with baseline (3.84 ± 0.49; P = .336 and P = .555, respectively). However, after serum dilution, the antibody titer nonsignificantly decreased 8-16 weeks after BTZ initiation (P = .345). Ten OIs were observed 24-72 weeks after BTZ initiation. CONCLUSIONS: Treatment with BTZ followed by cyclophosphamide yielded no significant decrease in antibody titer levels, and 10 OIs were observed during 24-72 weeks of BTZ treatment. No serious AEs were observed. Combining rituximab with BTZ is likely necessary to prevent generation of new autoantibody-producing plasma cells. Clinical Trials Registration. NCT03103555.

Ivermectin Accelerates Circulating Nonstructural Protein 1 (NS1) Clearance in Adult Dengue Patients: A Combined Phase 2/3 Randomized Double-blinded Placebo Controlled Trial.

BACKGROUND: Dengue is the most significant mosquito-borne viral disease; there are no specific therapeutics. The antiparasitic drug ivermectin efficiently inhibits the replication of all 4 dengue virus serotypes in vitro. METHODS: We conducted 2 consecutive randomized, double-blind, placebo-controlled trials in adult dengue patients to evaluate safety and virological and clinical efficacies of ivermectin. After a phase 2 trial with 2 or 3 days of 1 daily dose of 400 µg/kg ivermectin, we continued with a phase 3, placebo-controlled trial with 3 days of 400 µg/kg ivermectin. RESULTS: The phase 2 trial showed a trend in reduction of plasma nonstructural protein 1 (NS1) clearance time in the 3-day ivermectin group compared with placebo. Combining phase 2 and 3 trials, 203 patients were included in the intention to treat analysis (100 and 103 patients receiving ivermectin and placebo, respectively). Dengue hemorrhagic fever occurred in 24 (24.0%) of ivermectin-treated patients and 32 (31.1%) patients receiving placebo (P = .260). The median (95% confidence interval [CI]) clearance time of NS1 antigenemia was shorter in the ivermectin group (71.5 [95% CI 59.9-84.0] hours vs 95.8 [95% CI 83.9-120.0] hours, P = .014). At discharge, 72.0% and 47.6% of patients in the ivermectin and placebo groups, respectively had undetectable plasma NS1 (P = .001). There were no differences in the viremia clearance time and incidence of adverse events between the 2 groups. CONCLUSIONS: A 3-day 1 daily dose of 400 µg/kg oral ivermectin was safe and accelerated NS1 antigenemia clearance in dengue patients. However, clinical efficacy of ivermectin was not observed at this dosage regimen.

Epidemiology, clinical characteristics, and treatment outcomes of patients with COVID-19 at Thailand's university-based referral hospital.

BACKGROUND: The epidemiology and outcomes of COVID-19 patients in Thailand are scarce. METHODS: This retrospective cohort study included adult hospitalized patients who were diagnosed with COVID-19 at Siriraj Hospital during February 2020 to April 2020. RESULTS: The prevalence of COVID-19 was 7.5% (107 COVID-19 patients) among 1409 patients who underwent RT-PCR for SARS-CoV-2 detection at our hospital during the outbreak period. Patients with COVID-19 presented with symptoms in 94.4%. Among the 104 patients who were treated with antiviral medications, 78 (75%) received 2-drug regimen (lopinavir/ritonavir or darunavir/ritonavir plus chloroquine or hydroxychloroquine), and 26 (25%) received a 3-drug regimen with favipiravir added to the 2-drug regimen. Disease progression was observed in 18 patients (16.8%). All patients with COVID-19 were discharged alive. CONCLUSIONS: The prevalence of COVID-19 was 7.5% among patients who underwent RT-PCR testing, and 10% among those having risk factors for COVID-19 acquisition. Combination antiviral therapies for COVID-19 patients were well-tolerated and produced a favorable outcome.

The risk of Plasmodium vivax parasitaemia after P. falciparum malaria: An individual patient data meta-analysis from the WorldWide Antimalarial Resistance Network.

BACKGROUND: There is a high risk of Plasmodium vivax parasitaemia following treatment of falciparum malaria. Our study aimed to quantify this risk and the associated determinants using an individual patient data meta-analysis in order to identify populations in which a policy of universal radical cure, combining artemisinin-based combination therapy (ACT) with a hypnozoitocidal antimalarial drug, would be beneficial. METHODS AND FINDINGS: A systematic review of Medline, Embase, Web of Science, and the Cochrane Database of Systematic Reviews identified efficacy studies of uncomplicated falciparum malaria treated with ACT that were undertaken in regions coendemic for P. vivax between 1 January 1960 and 5 January 2018. Data from eligible studies were pooled using standardised methodology. The risk of P. vivax parasitaemia at days 42 and 63 and associated risk factors were investigated by multivariable Cox regression analyses. Study quality was assessed using a tool developed by the Joanna Briggs Institute. The study was registered in the International Prospective Register of Systematic Reviews (PROSPERO: CRD42018097400). In total, 42 studies enrolling 15,341 patients were included in the analysis, including 30 randomised controlled trials and 12 cohort studies. Overall, 14,146 (92.2%) patients had P. falciparum monoinfection and 1,195 (7.8%) mixed infection with P. falciparum and P. vivax. The median age was 17.0 years (interquartile range [IQR] = 9.0-29.0 years; range = 0-80 years), with 1,584 (10.3%) patients younger than 5 years. 2,711 (17.7%) patients were treated with artemether-lumefantrine (AL, 13 studies), 651 (4.2%) with artesunate-amodiaquine (AA, 6 studies), 7,340 (47.8%) with artesunate-mefloquine (AM, 25 studies), and 4,639 (30.2%) with dihydroartemisinin-piperaquine (DP, 16 studies). 14,537 patients (94.8%) were enrolled from the Asia-Pacific region, 684 (4.5%) from the Americas, and 120 (0.8%) from Africa. At day 42, the cumulative risk of vivax parasitaemia following treatment of P. falciparum was 31.1% (95% CI 28.9-33.4) after AL, 14.1% (95% CI 10.8-18.3) after AA, 7.4% (95% CI 6.7-8.1) after AM, and 4.5% (95% CI 3.9-5.3) after DP. By day 63, the risks had risen to 39.9% (95% CI 36.6-43.3), 42.4% (95% CI 34.7-51.2), 22.8% (95% CI 21.2-24.4), and 12.8% (95% CI 11.4-14.5), respectively. In multivariable analyses, the highest rate of P. vivax parasitaemia over 42 days of follow-up was in patients residing in areas of short relapse periodicity (adjusted hazard ratio [AHR] = 6.2, 95% CI 2.0-19.5; p = 0.002); patients treated with AL (AHR = 6.2, 95% CI 4.6-8.5; p < 0.001), AA (AHR = 2.3, 95% CI 1.4-3.7; p = 0.001), or AM (AHR = 1.4, 95% CI 1.0-1.9; p = 0.028) compared with DP; and patients who did not clear their initial parasitaemia within 2 days (AHR = 1.8, 95% CI 1.4-2.3; p < 0.001). The analysis was limited by heterogeneity between study populations and lack of data from very low transmission settings. Study quality was high. CONCLUSIONS: In this meta-analysis, we found a high risk of P. vivax parasitaemia after treatment of P. falciparum malaria that varied significantly between studies. These P. vivax infections are likely attributable to relapses that could be prevented with radical cure including a hypnozoitocidal agent; however, the benefits of such a novel strategy will vary considerably between geographical areas.

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.

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.).

Serological diagnosis of pulmonary Mycobacterium tuberculosis infection by LIPS using a multiple antigen mixture.

BACKGROUND: There is an urgent need for a simple and accurate test for the diagnosis of human Mycobacterium tuberculosis, the infectious agent causing tuberculosis (TB). Here we describe a serological test based on light emitting recombinant proteins for the diagnosis of pulmonary Mycobacterium tuberculosis infection. METHODS: Luciferase Immunoprecipitation Systems (LIPS), a fluid-phase immunoassay, was used to examine antibody responses against a panel of 24 different M. tuberculosis proteins. Three different strategies were used for generating the constructs expressing the recombinant fusion M. tuberculosis proteins with luciferase: synthetic gene synthesis, Gateway recombination cloning, and custom PCR synthesis. A pilot cohort of African pulmonary TB patients was used for initial antibody screening and confirmatory studies with selected antigens were performed with a cohort from Thailand and healthy US blood donors. In addition to testing M. tuberculosis antigens separately, a mixture that tested seven antigens simultaneously was evaluated for diagnostic performance. RESULTS: LIPS testing of a pilot set of serum samples from African pulmonary TB patients identified a potential subset of diagnostically useful M. tuberculosis antigens. Evaluation of a second independent cohort from Thailand validated highly significant antibody responses against seven antigens (PstS1, Rv0831c, FbpA, EspB, bfrB, HspX and ssb), which often showed robust antibody levels up to 50- to 1000-fold higher than local community controls. Marked heterogeneity of antibody responses was observed in the patients and the combined results demonstrated 73.5% sensitivity and 100% specificity for detection of pulmonary TB. A LIPS test simultaneously employing the seven M. tuberculosis antigen as a mixture matched the combined diagnostic performance of the separate tests, but showed an even higher diagnostic sensitivity (90%) when a cut-off based on healthy US blood donors was used. CONCLUSION: A LIPS immunoassay employing multiple M. tuberculosis antigens shows promise for the rapid and quantitative serological detection of pulmonary TB.

Anti-GM-CSF autoantibodies in patients with cryptococcal meningitis.

Cryptococcal meningitis has been described in immunocompromised patients, as well as in those for whom no immune defect has been identified. GM-CSF regulates the function of phagocytes and pulmonary alveolar macrophages, critical elements in cryptococcal control. We performed clinical histories, immunological evaluation, and anticytokine autoantibody screening in four current patients with cryptococcal meningitis and identified and tested 103 archived plasma/cerebrospinal fluid samples from patients with cryptococcal meningitis. We assessed the ability of anti-GM-CSF autoantibody-containing plasmas to inhibit GM-CSF signaling. We recognized anti-GM-CSF autoantibodies in an otherwise healthy female with cryptococcal meningitis who later developed pulmonary alveolar proteinosis (PAP). Her diagnosis prompted screening of patients with cryptococcal meningitis for anticytokine autoantibodies. We identified seven HIV-negative patients with cryptococcal meningitis who tested positive for high-titer anti-GM-CSF autoantibodies. Two of the seven later developed evidence of PAP. Plasma from all patients prevented GM-CSF-induced STAT5 phosphorylation and MIP-1α production in normal PBMCs. This effect was limited to their IgG fraction. Anti-GM-CSF autoantibodies are associated with some cases of cryptococcal meningitis in otherwise immunocompetent patients. These cases need not have associated PAP.

Prognostic indicators in adults hospitalized with falciparum malaria in Western Thailand.

BACKGROUND: Severe malaria remains a major cause of death and morbidity amongst adults in the Asiatic tropics. METHODS: A retrospective analysis of the clinical and laboratory data of 988 adult patients, hospitalized with Plasmodium falciparum malaria and prospectively recruited to malaria studies in western Thailand between 1986 and 2002, was performed to assess the factors associated with a fatal outcome. Different severity scores and classifications for defining severe malaria were compared and, using multiple logistic regression, simple models for predicting mortality developed. RESULTS: The proportion of patients fulfilling the WHO 2000 definition of severe malaria was 78.1%, and their mortality was 10%. Mortality in patients given parenteral artesunate or artemether (16/317, 5%) was lower than in those given parenteral quinine (59/442, 13%) (P < 0.001). Models using parameter sets based on WHO 1990, 2000 and Adapted AQ criteria plus blood smear parasite-stage assessment gave the best mortality prediction. A malaria prognostic index (MPI), derived from the dataset using five clinical or laboratory variables gave similar prognostic accuracy. CONCLUSIONS: The mortality of severe malaria in adults has fallen and the switch from quinine to artesunate has probably been an important contributor. Prognostic indices based on WHO 2000 definitions, and other simpler indices based on fewer variables, provide clinically useful predictions of outcome in Asian adults with severe malaria.

Fool's gold: Why imperfect reference tests are undermining the evaluation of novel diagnostics: a reevaluation of 5 diagnostic tests for leptospirosis.

BACKGROUND: We observed that some patients with clinical leptospirosis supported by positive results of rapid tests were negative for leptospirosis on the basis of our diagnostic gold standard, which involves isolation of Leptospira species from blood culture and/or a positive result of a microscopic agglutination test (MAT). We hypothesized that our reference standard was imperfect and used statistical modeling to investigate this hypothesis. METHODS: Data for 1652 patients with suspected leptospirosis recruited during three observational studies and one randomized control trial that described the application of culture, MAT, immunofluorescence assay (IFA), lateral flow (LF) and/or PCR targeting the 16S rRNA gene were reevaluated using Bayesian latent class models and random-effects meta-analysis. RESULTS: The estimated sensitivities of culture alone, MAT alone, and culture plus MAT (for which the result was considered positive if one or both tests had a positive result) were 10.5% (95% credible interval [CrI], 2.7%-27.5%), 49.8% (95% CrI, 37.6%-60.8%), and 55.5% (95% CrI, 42.9%-67.7%), respectively. These low sensitivities were present across all 4 studies. The estimated specificity of MAT alone (and of culture plus MAT) was 98.8% (95% CrI, 92.8%-100.0%). The estimated sensitivities and specificities of PCR (52.7% [95% CrI, 45.2%-60.6%] and 97.2% [95% CrI, 92.0%-99.8%], respectively), lateral flow test (85.6% [95% CrI, 77.5%-93.2%] and 96.2% [95% CrI, 87.7%-99.8%], respectively), and immunofluorescence assay (45.5% [95% CrI, 33.3%-60.9%] and 96.8% [95% CrI, 92.8%-99.8%], respectively) were considerably different from estimates in which culture plus MAT was considered a perfect gold standard test. CONCLUSIONS: Our findings show that culture plus MAT is an imperfect gold standard against which to compare alterative tests for the diagnosis of leptospirosis. Rapid point-of-care tests for this infection would bring an important improvement in patient care, but their future evaluation will require careful consideration of the reference test(s) used and the inclusion of appropriate statistical models.

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.

A Randomized Controlled Trial of Combined Ivermectin and Zinc Sulfate versus Combined Hydroxychloroquine, Darunavir/Ritonavir, and Zinc Sulfate among Adult Patients with Asymptomatic or Mild Coronavirus-19 Infection.

Introduction: Ivermectin, hydroxychloroquine (HQ), and darunavir/ritonavir are widely prescribed as an oral treatment for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection despite their uncertainty of clinical benefit. The objective is to determine the safety and the efficacies of two treatment regimens against SARS-CoV-2 infection. Methods: We conducted an open-labeled, randomized, controlled trial to compare the efficacy between a 3-day course of once-daily high-dose oral ivermectin plus zinc sulfate (Group A) and a combination of HQ, darunavir/ritonavir, and zinc sulfate (HQ + antiretroviral, Group B) for 5 days in asymptomatic or mild SARS-CoV-2 infection. The study period was between December 2020 and April 2021. Results: Overall, 113 patients were randomized and analyzed (57 patients in Group A and 56 patients in Group B). The median duration to achieve the virological outcome of either undetected or cycle threshold (Ct) for N gene of SARS-CoV-2 by real-time polymerase chain reaction was 6 days (95% confidence interval [CI] 5.3-6.7) versus 7 days (95% CI: 5.4-8.6) in Group A and Group B, respectively (P = 0.419) in the modified intention-to-treat population. All patients were discharged from hospital quarantine as planned. Two patients in Group A and one patient in Group B were considered clinically worsening and received 10 days of favipiravir treatment. There was no serious adverse event found in both groups. Conclusion: We demonstrated that both treatment regimens were safe, but both treatment regimens had no virological or clinical benefit. Based on this result and current data, there is no supporting evidence for the clinical benefit of ivermectin for coronavirus-19.

The Abbott Pandemic Defense Coalition: a unique multisector approach adds to global pandemic preparedness efforts.

Detection and epidemiologic characterization of infectious disease outbreaks are key for early identification and response to potential pandemic threats. The rapid global spread of severe SARS-CoV-2 in 2020 highlighted the critical role of diagnostics in understanding the epidemiology of the virus early in the pandemic. As a natural extension of Abbott's work in diagnostics, virus discovery, and virus surveillance, the Abbott Pandemic Defense Coalition (APDC) was launched in early 2021. The APDC is a global multisector scientific and public health partnership whose primary objective is the early detection and mitigation of infectious disease threats of pandemic potential. As of January 2022, the APDC network has partners on 5 continents including academic institutions, governmental, and nongovernmental organizations. A novel element of the APDC is the capacity for early development and rapid deployment of scalable, quality diagnostics targeting newly identified pathogens of pandemic potential.

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.

Leptospira collagenase and LipL32 for antibody detection in leptospirosis.

Various Leptospira components have been identified as candidate antigens for the detection of antibody to Leptospira. LipL32 is a Leptospira membrane protein which has been widely studied. The report of Leptospira whole-genome sequencing demonstrated that pathogenic Leptospira contained the nucleotide sequence (colA gene) coding for the collagenase. Expression of ColA protein and its enzymatic activity was demonstrated. In this study, cloned ColA protein, in comparison with LipL32, was used as an antigen for antibody detection. Thirty pairs of sera from human leptospirosis patients were tested. Sera from blood donors, and patients with scrub typhus and dengue virus infection (20 samples from each group) were tested for the specificity. All sera from leptospirosis patients tested in this study reacted with both ColA and LipL32 proteins. Sera from blood donors, patients with scrub typhus and dengue virus infection did not react with ColA protein. Data suggested that sensitivity and specificity of ColA protein for Leptospira antibody detection were 100%. In addition, ColA protein showed higher specificity than LipL32. Our data suggested that ColA protein could be another candidate antigen for antibody detection in leptospirosis diagnosis.

Comparative genome characterization of Leptospira interrogans from mild and severe leptospirosis patients.

Leptospirosis is a zoonotic disease caused by spirochetes from the genus Leptospira. In Thailand, Leptospira interrogans is a major cause of leptospirosis. Leptospirosis patients present with a wide range of clinical manifestations from asymptomatic, mild infections to severe illness involving organ failure. For better understanding the difference between Leptospira isolates causing mild and severe leptospirosis, illumina sequencing was used to sequence genomic DNA in both serotypes. DNA of Leptospira isolated from two patients, one with mild and another with severe symptoms, were included in this study. The paired-end reads were removed adapters and trimmed with Q30 score using Trimmomatic. Trimmed reads were constructed to contigs and scaffolds using SPAdes. Cross-contamination of scaffolds was evaluated by ContEst16s. Prokka tool for bacterial annotation was used to annotate sequences from both Leptospira isolates. Predicted amino acid sequences from Prokka were searched in EggNOG and David gene ontology database to characterize gene ontology. In addition, Leptospira from mild and severe patients, that passed the criteria e-value < 10e-5 from blastP against virulence factor database, were used to analyze with Venn diagram. From this study, we found 13 and 12 genes that were unique in the isolates from mild and severe patients, respectively. The 12 genes in the severe isolate might be virulence factor genes that affect disease severity. However, these genes should be validated in further study.

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.

Case Report: Recovery of Pathogenic Leptospira spp. from Routine Aerobic Blood Culture Bottles.

Leptospira spp. are fastidious and slow-growing bacteria, making recovery difficult and diagnostic sensitivity in the clinical setting low. However, collection of Leptospira isolates is valuable for epidemiological and laboratory research. Severe leptospirosis cases may present as septic shock, and the differential diagnosis often includes bacterial septicemia, leading clinicians to collect blood cultures. Here, we report the successful isolation of pathogenic Leptospira spp. from blood culture bottles (targeting aerobic bacteria incubated at 37°C) from a 64-year-old man admitted with septic shock. The patient presented with 4 days of fever, severe hypotension, transient atrial fibrillation, jaundice, and oliguric renal failure. After admission, intravenous ceftriaxone plus azithromycin was given with fluid resuscitation, norepinephrine infusion, invasive mechanical ventilation, and renal replacement therapy. He was discharged from the hospital 16 days after admission. Using the blood sample obtained on admission, the diagnosis of leptospirosis was confirmed by multiplex real-time PCR (targeting bacterial 16S rRNA and LipL32 gene). We collected 200 µL from the blood culture bottle to inoculate a 5-mL Ellinghausen, McCullough, Johnson, and Harris media supplemented with 5% fetal bovine serum. After 2 weeks of incubation at 30°C, Leptospira strains were identified and confirmed by real-time PCR. Genotyping was undertaken using the multi-locus sequence typing (MLST) scheme#1. The isolate matched with ST50 isolates in the PUbMLST database. This case provides evidence that in tropical countries, severe leptospirosis should be considered in patients who present with symptoms of sepsis. Pathogenic Leptospira may be successfully isolated from aerobic blood cultures in routine clinical settings.