Shared and unique antibody and B cell profiles in HIV-positive and HIV-negative individuals with cryptococcal meningoencephalitis.

Host non-T cell markers to aid in the diagnosis of cryptococcal meningoencephalitis (CM) have not been identified. In this case-control study, we characterized antibody and B cell profiles in HIV-negative and HIV-positive Vietnamese individuals of the Kinh ethnicity recently diagnosed with CM and controls. The study included 60 HIV-negative with no known immunocompromising condition and 60 HIV-positive individuals, with 30 CM cases and 30 controls in each group. Participants were matched by age, sex, HIV serostatus, and CD4 count in the HIV-positive group. Plasma immunoglobulin (Ig) levels, including IgG1, IgG2, IgM, and IgA, Cryptococcus spp. glucuronoxylomannan (GXM)- and laminarin (branched ${\rm{\beta }}$-[1-3]-glucan)-binding IgG, IgM, IgA levels, and peripheral blood B cell subsets were measured. Logistic regression, principal component, and mediation analyses were conducted to assess associations between antibody, B cell levels, and CM. The results showed that GXM-IgG levels were higher and IgG1 and IgG2 were lower in CM cases than controls, regardless of HIV status. In HIV-negative individuals, IgG2 mediated an inverse association between CD19+CD27+CD43+CD5- (B-1b-like) cells and CM. In HIV-positive individuals, lower levels of IgA, laminarin-IgA, and CD19+CD27+IgM+IgD- (IgM+ memory B) cells were each associated with CM. The shared and distinct antibody and B cell profiles identified in HIV-negative and HIV-positive CM cases may inform the identification of non-T-cell markers of CM risk or unsuspected disease, particularly in HIV-negative individuals.

Unlike cryptococcal meningitis (CM) in HIV-positive individuals, there are no known biomarkers of risk in HIV-negative individuals and the diagnosis is often not suspected and delayed. This study identified non-T cells, including antibody and B cell CM-associated profiles that may guide cryptococcal antigen testing in HIV-negative individuals.

An open label randomized controlled trial of tamoxifen combined with amphotericin B and fluconazole for cryptococcal meningitis.

Background: Cryptococcal meningitis has high mortality. Flucytosine is a key treatment but is expensive and rarely available. The anticancer agent tamoxifen has synergistic anti-cryptococcal activity with amphotericin in vitro. It is off-patent, cheap, and widely available. We performed a trial to determine its therapeutic potential. Methods: Open label randomized controlled trial. Participants received standard care - amphotericin combined with fluconazole for the first 2 weeks - or standard care plus tamoxifen 300 mg/day. The primary end point was Early Fungicidal Activity (EFA) - the rate of yeast clearance from cerebrospinal fluid (CSF). Trial registration https://clinicaltrials.gov/ct2/show/NCT03112031. Results: Fifty patients were enrolled (median age 34 years, 35 male). Tamoxifen had no effect on EFA (-0.48log10 colony-forming units/mL/CSF control arm versus -0.49 tamoxifen arm, difference -0.005log10CFU/ml/day, 95% CI: -0.16, 0.15, p=0.95). Tamoxifen caused QTc prolongation. Conclusions: High-dose tamoxifen does not increase the clearance rate of Cryptococcus from CSF. Novel, affordable therapies are needed. Funding: The trial was funded through the Wellcome Trust Asia Programme Vietnam Core Grant 106680 and a Wellcome Trust Intermediate Fellowship to JND grant number WT097147MA.

A randomized open label trial of tamoxifen combined with amphotericin B and fluconazole for cryptococcal meningitis.

Background: Cryptococcal meningitis is a leading cause of death in HIV-infected patients. International treatment guidelines recommend induction therapy with amphotericin B and flucytosine. This antifungal combination is most effective, but unfortunately flucytosine is expensive and unavailable where the burden of disease is greatest. Where unavailable, guidelines recommend treatment with amphotericin and fluconazole, but this is less effective, with mortality rates of 40-50%. Faster rates of clearance of yeast from cerebrospinal fluid (CSF) are associated with better outcomes - improving the potency of antifungal therapy is likely to be an effective strategy to improve survival. Tamoxifen, a selective estrogen receptor modulator used to treat breast cancer, has anti-cryptococcal activity, appearing synergistic when combined in vitro with amphotericin, and fungicidal when combined with fluconazole. It is concentrated in the brain and macrophages, off-patent, cheap and widely available. We designed a randomized trial to deliver initial efficacy and safety data for tamoxifen combined with amphotericin and fluconazole. Method: A phase II, open-label, randomized (1:1) controlled trial of tamoxifen (300mg/day) combined with amphotericin (1mg/kg/day) and fluconazole (800mg/day) for the first 2 weeks therapy for HIV infected or uninfected adults with cryptococcal meningitis. The study recruits at Cho Ray Hospital and the Hospital for Tropical Diseases, Ho Chi Minh City, Vietnam. The primary end point is Early Fungicidal Activity (EFA-the rate of yeast clearance from CSF), over the first two weeks of treatment. 50 patients will be recruited providing ≈80% and 90% power to detect a difference in the EFA of -0.11 or -0.13 log10CFU/ml/day, respectively. Discussion: The results of the study will inform the decision to proceed to a larger trial powered to mortality. The size of effect detectable has previously been associated with reduced mortality from this devastating disease. Particular side effects of interest include QT prolongation. Trial registration: Clinicaltrials.gov NCT03112031 (11/04/2017).

Antiproliferative activity and apoptosis induction by trijuganone C isolated from the root of Salvia miltiorrhiza Bunge (Danshen).

In this study, we found that the hexane fraction of Danshen, the dried root of Salvia miltiorrhiza (Lamiaceae), exerted antiproliferative effects on human leukemia cells. Phytochemical investigation of the hexane fraction achieved the isolation of the tanshinone diterpenes: dihydrotanshinone I (1), trijuganone C (2), trijuganone B (3), cryptotanshinone (4), tanshinone IIA (5), and tanshinone I (6). Compound 2 showed significant antiproliferative activities against human leukemia cells HL-60, Jurkat, and U937. The antiproliferative activities of 2 against human cancer and normal cells indicated that 2 exhibited potent antiproliferative activities with IC50 values less than 10 µM against HL-60 and Jurkat cells as well as on the colon cancer cells DLD-1, COLO 205, and Caco-2. Compound 2 induced chromatin condensation, DNA fragmentation, activation of caspase-3, -8, and -9, and the cleavage of poly (ADP-ribose) polymerase (PARP) in HL-60 cells. Moreover, 2 activated Bid and Bax, leading to the loss of mitochondrial membrane potential, and 2 induced the cytochrome c release from mitochondria into cytosol. In contrast, Bcl-2 and Bcl-xL were unaffected by 2. These results suggest that 2 exerts antiproliferative effects via apoptosis induction mediated by mitochondrial dysfunction and caspase activation. Compound 2 may serve as a candidate of potential chemotherapeutic agent for human leukemia.

Spirally oriented Au microelectrode array sensor for detection of Hg (II).

A simple and reproducible carbon microelectrode array (CMA), designed to eliminate diffusive interference among the microelectrodes, has been fabricated and used as a frame to build a gold (Au) microelectrode array (GMA) sensor. To prepare the CMA initially, rather than use an uncontrollable large number of carbon fibers, only 60 carbon fibers of regular size were used to ensure manageable and reproducible arrangement for array construction. In addition, for efficient spatial arrangement of the microelectrode and easy sensor preparation, carbon fibers were oriented in a spiral fashion by rolling around a Cu wire. The distance between carbon fibers was carefully determined to avoid overlap among individual diffusion layers, one of the important factors governing steady-state current response and sensor-to-sensor reproducibility. After the preparation of a spirally arranged CMA, Au was electrochemically deposited on the surface of individual carbon electrodes to build a final GMA sensor. Then, the GMA sensor was used to measure Hg(2+) in a low concentration range. Simultaneously, multiple GMA sensors were independently prepared to examine reproducibility in sensor fabrication as well as electrochemical measurement (sensor-to-sensor reproducibility). Overall, highly sensitive detection of Hg(2+) was possible using the proposed GMA sensor due to efficient arrangement of microelectrodes and the sensor-to-sensor reproducibility was superior owing to simplicity in sensor fabrication.

Clinical, epidemiological and virological features of Dengue virus infections in Vietnamese patients presenting to primary care facilities with acute undifferentiated fever.

OBJECTIVES: To explore clinical and virological characteristics and describe the epidemiology of dengue in patients who presented with acute undifferentiated fever (AUF) at primary health centers (PHC) in Binh Thuan Province, Vietnam. METHODS: A prospective observational study was conducted from 2001 to 2006 to study the aetiology in AUF patients. Demographic and clinical information was obtained, and dengue polymerase chain reaction (RT-PCR) and serology were performed on a random selection of patients. RESULTS: Three hundred fifty-one serologically confirmed dengue patients including 68 primary and 283 secondary infections were included in this study. In 25% (86/351) dengue virus (DENV) was detected by RT-PCR among which 32 DENV-1, 16 DENV-2, 1 DENV-3 and 37 DENV-4 were identified. The predominant dengue serotype varied by year with seasonal fluctuation: DENV-4 in 2001-2002, DENV-1 and DENV-2 from 2003 to 2006. Primary dengue was more common in children. Higher viraemia levels (P=0.010) were found in primary infections compared to secondary infections. DENV-1 infected patients had higher viraemia levels than DENV-2 (P=0.003) and DENV-4 (P<0.001) infected patients. Clinical symptoms were often seen in adults. Few differences in clinical symptoms were found between primary and secondary infection and no significant differences in clinical symptoms between the serotypes were observed. CONCLUSIONS: Our data provide insight in the epidemiology, clinical profile and virological features of mild symptomatic dengue patients who presented to PHC with AUF in Vietnam.

Square wave voltammetric detection of Anthrax utilizing a peptide for selective recognition of a protein biomarker.

A short chain peptide (16mer) has been successfully utilized for the selective electrochemical detection of the protein biomarker, protective antigen (PA), for the diagnosis of Anthrax. The major motivation of using a peptide instead of an antibody for the development of a biosensor is that there are advantages associated with the smaller size, better biological stability and easy synthesizability of a peptide. PA-selective peptide was synthesized and conjugated on a binding layer previously immobilized onto gold electrode. The same sensing scheme using a conventional antibody instead of a peptide was also tested for the purpose of comparison. Since the size of the peptide is approximately 1-2 orders of magnitude smaller than that of the antibody, a more populated immobilization of peptide on the sensing layer is possible and will eventually lead to improved sensitivity for PA detection. The selectivity of the peptide-based sensor was evaluated by observing the reduction peaks of samples containing PA with different concentrations of bovine serum albumin (BSA). The resulting responses were interference-free from BSA. The results of this study demonstrate the strong analytical potential of a peptide-based biosensor for diverse applications, especially in disease diagnosis where detection of a specific protein biomarker is particularly demanding.

Seroepidemiology and serological follow-up of anti-leptospiral IgG in children in Southern Vietnam.

A follow-up study was conducted with 23 months interval to investigate the seroepidemiology and persistence of Leptospira IgG antibodies among healthy children in Binh Thuan province, Southern Vietnam. Sera from 262 children (7-13 years of age) were collected and analysed with a commercially available enzyme-linked immunosorbent assay (ELISA) for Leptospira IgG. Seroconversion was observed in 10.4% (22 of 211, 95% CI: 5.6-26.7) of the children, of whom 18 (8.5%) had probably and four (1.9%) had certainly been exposed to Leptospira. Based on the reduction of sero-negatives of 1.9% among children who have been certainly exposed, the annual seroconversion rate, a measure of the incidence rate of Leptospira infections, corresponds to 0.99% (95% CI: 0.39-2.52). In 61% (31 of 51, 95% CI: 47.1-73.0) of the children with past-infection, Leptospira IgG antibodies remain detectable after 2 years. Data from this study indicate that IgG antibody responses against Leptospira may persist at least for 2 years in children without manifestations of leptospirosis. Results of study uncover the true incidence of leptospirosis infection, the dynamics of waxing and waning antibody concentrations and points at a larger burden of clinically non-significant Leptospira infections in Southern Vietnam. This also indicates background reactivity for serological testing and thus serological result of a single serum sample must be carefully interpreted.

Incidence of primary dengue virus infections in Southern Vietnamese children and reactivity against other flaviviruses.

OBJECTIVE: To study the incidence of asymptomatic primary dengue infections among children and reactivity against other flaviviruses. METHODS: A total of 216 children, who had no dengue-specific IgG antibodies during a serosurvey in 2003 were re-examined 23 months later to determine if seroconversion had occurred. Dengue-specific IgG was demonstrated with enzyme-linked immunosorbent assay (ELISA) and reactivity patterns against other flaviviruses were assessed by using immunofluorescence assay (IFA). RESULTS: Sixty-six children had seroconverted for dengue virus-specific IgG; the true annual incidence of primary dengue was thus 17.3% (95% CI: 13.8-21.4). Japanese Encephalitis virus (JEV)-specific IgG antibodies were detected by IFA among three (4.6%) samples that showed seroconversion in the dengue ELISA, because of cross-reactivity. CONCLUSION: Our findings highlight the high incidence of dengue among Vietnamese children; JEV infections are rare. The true annual incidence of dengue can be estimated with a single cross-sectional seroprevalence survey.

Evaluation of two rapid immunochromatographic assays for diagnosis of dengue among Vietnamese febrile patients.

Results from two dengue rapid tests, the PanBio Duo cassette and the SD Bioline strip test, were compared to those of enzyme-linked immunosorbent assays (Focus Diagnostics) from sera of 200 Vietnamese febrile patients. The PanBio assay was superior, with sensitivity and specificity values for acute-phase serum samples of 54% and 70% (immunoglobulin M) and 70% and 88% (immunoglobulin G), respectively.

Seroepidemiology of leptospirosis in southern Vietnamese children.

OBJECTIVE: To estimate the seroprevalence of human leptospirosis in southern Vietnam. METHODS: All pupils (n=961) of two primary schools in two communes in southern Vietnam were screened for the presence of serum Leptospira immunoglobulin (Ig)G. Leptospira IgM was tested in 92 randomly selected samples. IgM and IgG were tested with a commercially available enzyme-linked immunosorbent assay (ELISA). RESULTS: Leptospira IgG was found in 123 (12.8%, 95% CI: 10.8-15.1) children with a male:female ratio of 1.5:1. At the age of 7 years the prevalence was 11%. Log-linear binary regression of the IgG seroprevalence by age showed a mean seroconversion rate of 1.5% (95% CI: 1.23-1.75) per year. Five (5.4%, 95% CI: 2.3-12.1) samples were definitely positive for IgM. Presence of serum Leptospira IgG antibodies was significantly associated with swimming in rivers (RR: 1.745, 95% CI: 1.117-2.724) and wading through water (RR 1.793, 95% CI: 1.181-2.722). CONCLUSION: Our data emphasize the importance of leptospirosis among children in Vietnam, despite the absence of severe disease, and stress the need for adequate and cheap diagnostics.

Seroprevalence of dengue antibodies, annual incidence and risk factors among children in southern Vietnam.

Dengue is highly endemic in southern Vietnam and all four serotypes of dengue virus have already been identified. To determine the age-specific prevalence of dengue and associated risk factors, we conducted a serological study at two primary schools and assessed risk factors by analysing children's questionnaires and household surveys. Sera were collected from 961 primary schoolchildren in Binh Thuan Province and tested for the presence of dengue virus serum antibodies using an indirect immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA). The antibody prevalence of the total population was 65.7% (n=631) which increased from 53.0 to 88.2% with age. The annual incidence of a first dengue infection, estimated by binary regression of the seroprevalence by age, was 11.7%. Interestingly, the prevalence of dengue IgG antibodies was significantly higher in children who confirmed using a pit latrine (RR 1.467, 95% CI: 1.245-1.730) and whose domestic environment contained discarded cans (RR 1.238, 95% CI: 1.042-1.470) and pigs (RR 1.228, 95% CI: 1.002-1.504). The epidemiology of dengue in southern Vietnam is stable with a constantly high annual incidence of first infections. Transmission occurs mainly peri-domestically, which has important public health implications.