Urine Levels of Defensin α1 Reflect Kidney Injury in Leptospirosis Patients.

Leptospirosis is a zoonotic disease whose severe forms are often accompanied by kidney dysfunction. In the present study, urinary markers were studied for potential prediction of disease severity. Urine samples from 135 patients with or without leptospirosis at San Lazaro Hospital, the Philippines, were analyzed. Urine levels of defensin α1 (uDA1) were compared with those of neutrophil gelatinase-associated lipocalin (uNGAL) and N-acetyl-ß-d-glucosidase (uNAG). Serum creatinine (Cr) was used as a marker of kidney injury. The levels of uDA1/Cr, uNGAL/Cr, and uNAG/Cr were positive in 46%, 90%, and 80% of leptospirosis patients, and 69%, 70%, and 70% of non-leptospirosis patients, respectively. In leptospirosis patients, the correlation of uDA1/Cr, uNGAL/Cr and uNAG/Cr levels with serum Cr were r = 0.3 (p < 0.01), r = 0.29 (p < 0.01), and r = 0.02 (p = 0.81), respectively. uDA1/Cr levels were correlated with uNGAL/Cr levels (r = 0.49, p < 0.01) and uNAG/Cr levels (r = 0.47, p < 0.0001) in leptospirosis patients. These findings suggest that uDA1, uNGAL, and uNAG were elevated in leptospirosis patients and reflected various types of kidney damage. uDA1 and uNGAL can be used to track kidney injury in leptospirosis patients because of their correlation with the serum Cr level.

Combined antibody and DNA detection for early diagnosis of leptospirosis after a disaster.

Early diagnosis based on laboratory confirmation is essential for managing leptospirosis. This study investigated the effectiveness of a novel method of detecting leptospirosis that combines measurement of anti-Leptospira antibodies by the microscopic agglutination test (MAT), enzyme-linked immunosorbent assay (ELISA), and immunochromatographic test (ICT) and leptospiral DNA by loop-mediated isothermal amplification (LAMP) and real-time PCR in plasma and 2 types of urine pellets. Of 113 suspected cases, 68.1%, 76.1%, and 60.2% were positive by MAT, ELISA, and ICT, respectively. Real-time PCR using DNA purified from urine pellets collected by low-speed centrifugation yielded positive signals for patients in late acute as well as early phase who were positive by LAMP using plasma DNA or urine pellets. Among antibody-negative patients, 9.5% were positive by DNA detection. These findings indicate that the leptospirosis detection rate is increased by combining antibody and DNA detection, providing a new tool for timely diagnosis of infection.

Elevated levels of full-length and thrombin-cleaved osteopontin during acute dengue virus infection are associated with coagulation abnormalities.

INTRODUCTION: Dengue virus (DENV) is transmitted by the mosquito vector, and causes a wide range of symptoms that lead to dengue fever (DF) or life-threatening dengue hemorrhagic fever (DHF). The host and viral correlates that contribute to DF and DHF are complex and poorly understood, but appear to be linked to inflammation and impaired coagulation. Full-length osteopontin (FL-OPN), a glycoprotein, and its activated thrombin-cleaved product, trOPN, integrate multiple immunological signals through the induction of pro-inflammatory cytokines. MATERIALS AND METHOD: To understand the role of OPN in DENV-infection, we assessed circulating levels of FL-OPN, trOPN, and several coagulation markers (D-dimer, thrombin-antithrombin complex [TAT], thrombomodulin [TM], and ferritin in blood obtained from 65 DENV infected patients in the critical and recovery phases of DF and DHF during a dengue virus epidemic in the Philippines in 2010. RESULTS: Levels of FL-OPN, trOPN, D-dimer, TAT, and TM were significantly elevated in the critical phase in both the DF and DHF groups, as compared with healthy controls. During the recovery phase, FL-OPN levels declined while trOPN levels increased dramatically in both the DF and DHF groups. FL-OPN levels were directly correlated with D-dimer and ferritin levels, while the generation of trOPN was associated with TAT levels, platelet counts, and viral RNA load. CONCLUSION: Our study demonstrated the marked elevation of plasma levels of FL-OPN and thrombin-cleaved OPN product, trOPN, in DENV-infection for the first time. Further studies on the biological functions of these matricellular proteins in DENV-infection would clarify its pathogenesis.

Galectin-9 plasma levels reflect adverse hematological and immunological features in acute dengue virus infection.

BACKGROUND: Dengue virus (DENV) infection remains a major public health burden worldwide. Soluble mediators may play a critical role in the pathogenesis of acute DENV infection. Galectin-9 (Gal-9) is a soluble ß-galactoside-binding lectin, with multiple immunoregulatory and inflammatory properties. OBJECTIVE: To investigate plasma Gal-9 levels as a biomarker for DENV infection. STUDY DESIGN: We enrolled 65 DENV infected patients during the 2010 epidemic in the Philippines and measured their plasma Gal-9 and cytokine/chemokine levels, DENV genotypes, and copy number during the critical and recovery phases of illness. RESULTS: During the critical phase, Gal-9 levels were significantly higher in DENV infected patients compared to healthy or those with non-dengue febrile illness. The highest Gal-9 levels were observed in dengue hemorrhagic fever (DHF) patients (DHF: 2464 pg/ml; dengue fever patients (DF): 1407 pg/ml; non-dengue febrile illness: 616 pg/ml; healthy: 196 pg/ml). In the recovery phase, Gal-9 levels significantly declined from peak levels in DF and DHF patients. Gal-9 levels tracked viral load, and were associated with multiple cytokines and chemokines (IL-1α, IL-8, IP-10, and VEGF), including monocyte frequencies and hematologic variables of coagulation. Further discriminant analyses showed that eotaxin, Gal-9, IFN-α2, and MCP-1 could detect 92% of DHF and 79.3% of DF, specifically (P<0.01). CONCLUSION: Gal-9 appears to track DENV inflammatory responses, and therefore, it could serve as an important novel biomarker of acute DENV infection and disease severity.

Possible HIV transmission modes among at-risk groups at an early epidemic stage in the Philippines.

A concentrated human immunodeficiency virus (HIV) epidemic might have started in the Philippines. A subsequent characterization of viruses was carried out to estimate HIV transmission modes. Most HIV strains from injecting drug users belonged to subtype-B. CRF-01 was a major subtype harbored by three other at-risk populations: male visa applicants who had sex with men, "men who have sex with men," and visa applicants. An HIV phylogeny suggested that two strain groups of injecting drug users and others circulated separately. In contrast, there was substantial genetic overlap between two strain groups from "men who have sex with men" and visa applicants. Mean nucleotide distance within strains was shorter among subtype-B strains harbored by the injecting drug users (0.010) than among CRF-01 strains of the other three populations: male visa applicants who had sex with men (0.034), "men who have sex with men" (0.023), and visa applicants (0.032). Closely related strains of hepatitis C virus were derived from not only HIV-positive but also -negative individuals. These results suggest that there is potential for transmission from visa applicants to "men who have sex with men," and that once HIV occurs in injecting drug users, it spreads rapidly among them. Close contacts of hepatitis C virus carriers composed of HIV-negative and -positive individuals indicated ongoing HIV spread via blood and possible intervention points. Large-scale analysis is needed to provide more precise information on the transmission directions and to help curb the growth of this HIV epidemic in the Philippines.

Frequent detection of anti-tubercular-glycolipid-IgG and -IgA antibodies in healthcare workers with latent tuberculosis infection in the Philippines.

Anti-tubercular-glycolipid-IgG (TBGL-IgG) and -IgA (TBGL-IgA) antibodies, and the QuantiFERON-TB Gold test (QFT) were compared in healthcare workers (HCWs, n = 31) and asymptomatic human immunodeficiency virus-carriers (HIV-AC, n = 56) in Manila. In HCWs, 48%, 51%, and 19% were positive in QFT, TBGL-IgG, and -IgA, respectively. The TBGL-IgG positivity was significantly higher (P = 0.02) in QFT-positive than QFT-negative HCWs. Both TBGL-IgG- and -IgA-positive cases were only found in QFT-positive HCWs (27%). The plasma IFN-γ levels positively correlated with TBGL-IgA titers (r = 0.74, P = 0.005), but not TBGL-IgG titers in this group, indicating that mucosal immunity is involved in LTBI in immunocompetent individuals. The QFT positivity in HIV-AC was 31% in those with CD4+ cell counts >350/µL and 12.5% in low CD4 group (<350/µL). 59 % and 29% were positive for TBGL-IgG and -IgA, respectively, in HIV-AC, but no association was found between QFT and TBGL assays. TBGL-IgG-positive rates in QFT-positive and QFT-negative HIV-AC were 61% and 58%, and those of TBGL-IgA were 23% and 30%, respectively. The titers of TBGL-IgA were associated with serum IgA (P = 0.02) in HIV-AC. Elevations of TBGL-IgG and -IgA were related to latent tuberculosis infection in HCWs, but careful interpretation is necessary in HIV-AC.

The early phase of an HIV epidemic in a population exposed previously to HCV in the Philippines.

Human immunodeficiency virus (HIV) sentinel surveillance program for injecting drug users has been conducted in Metro Cebu, the Philippines. A low prevalence (0-0.52%) of anti-HIV-positivity had been detected in this population from 2002 to 2007. However, a 10-fold increase in HIV prevalence was detected in the 2009 national HIV sentinel surveillance program. It prompted an additional outreach program to be conducted in Metro Cebu in January 2010, which recorded the highest HIV prevalence rate ever documented in the Philippines (75%, 44/59). HIV genes from fourteen 2009 to 2010 Metro Cebu strains were clustered closely in the phylogenetic tree, but no other strain collected outside Metro Cebu and none stored in the International Nucleotide Sequence Database was allocated to the same phylogenetic cluster. All these HIV infections have emerged in the anti-hepatitis C virus (HCV)-positive population (100%, 62/62) in Metro Cebu from 2009 to 2010. The five HCV strains from the individuals harboring the closely related HIV strains were categorized into different subtypes. These results strongly suggest that HIV infections occurred recently and spread rapidly among injecting drug users, while HCV had been circulating previously among them. Considering the fact that injecting drug use was the first mode of HIV transmission in Asia, extensive monitoring of injecting drug users and associated bridging populations is necessary. Therefore, HCV-guided characterization of the spread of HIV to populations that are vulnerable to blood-borne infections could play an important role in alerting health authorities to the early phase of an HIV epidemic.

Multi-country evaluation of the sensitivity and specificity of two commercially-available NS1 ELISA assays for dengue diagnosis.

BACKGROUND: Early diagnosis of dengue can assist patient triage and management and prevent unnecessary treatments and interventions. Commercially available assays that detect the dengue virus protein NS1 in the plasma/serum of patients offers the possibility of early and rapid diagnosis. METHODOLOGY/PRINCIPAL FINDINGS: The sensitivity and specificity of the Pan-E Dengue Early ELISA and the Platelia Dengue NS1 Ag assays were compared against a reference diagnosis in 1385 patients in 6 countries in Asia and the Americas. Platelia was more sensitive (66%) than Pan-E (52%) in confirmed dengue cases. Sensitivity varied by geographic region, with both assays generally being more sensitive in patients from SE Asia than the Americas. Both kits were more sensitive for specimens collected within the first few days of illness onset relative to later time points. Pan-E and Platelia were both 100% specific in febrile patients without evidence of acute dengue. In patients with other confirmed diagnoses and healthy blood donors, Platelia was more specific (100%) than Pan-E (90%). For Platelia, when either the NS1 test or the IgM test on the acute sample was positive, the sensitivity versus the reference result was 82% in samples collected in the first four days of fever. NS1 sensitivity was not associated to disease severity (DF or DHF) in the Platelia test, whereas a trend for higher sensitivity in DHF cases was seen in the Pan-E test (however combined with lower overall sensitivity). CONCLUSIONS/SIGNIFICANCE: Collectively, this multi-country study suggests that the best performing NS1 assay (Platelia) had moderate sensitivity (median 64%, range 34-76%) and high specificity (100%) for the diagnosis of dengue. The poor sensitivity of the evaluated assays in some geographical regions suggests further assessments are needed. The combination of NS1 and IgM detection in samples collected in the first few days of fever increased the overall dengue diagnostic sensitivity.

Tracking the entry routes of hepatitis C virus as a surrogate of HIV in an HIV-low prevalence country, the Philippines.

From 2002 to 2007, 1,590 individuals were enrolled in an active surveillance program conducted in Metro Cebu, Philippines, where the anti-HCV-positive rate was significantly and constantly high among injecting drug users (83%, 793/960; 71-88%), especially among those living in downtown (89%, 683/770; 87-100%), despite the extremely low percentage of anti-HIV-positives (0.34%, 3/874). Sampling areas were then enlarged nationwide and the number of samples increased to 2,645 at the end of 2007. A total of 444 samples were positive for HCV RNA. Phylogenetic analysis based on NS5B and E1-E2 regions revealed that the most dominant HCV subtype was 1a, and followed by 2b, 2a, and 1b, and that the HCV strains had the largest variety in Metro Manila and its vicinity (P < 0.01). Interestingly, subtype 1b was detected solely in Metro Manila, and four HCV strains collected in this area showed higher homology to specific foreign strains retrieved from the Genbank/EMBL/DDBJ database with bootstrap values of 68-95% comparing with other strains analyzed in this nationwide study. These data suggest that HCV strains may be introduced occasionally into the Philippines possibly through Metro Manila as a main entry point. Considering the fact that an HIV epidemic started primarily via contaminated needle sharing in Asia, the constantly high rate of HCV infections and the newly introduced foreign HCV strains in the absence of HIV epidemic warrant further investigation on HCV entry and spread for early detection of an HIV epidemic in the Philippines.

The prevalence of HIV, HBV and HCV among Filipino blood donors and overseas work visa applicants.

OBJECTIVE: Our aim was to estimate the prevalence of HIV, HBV and HCV among the general population of the Philippines using data sources outside of the limited existing active surveillance network. METHODS: We analysed aggregate HIV, HBV and HCV test results for hospital-based blood donors (BDs) and overseas Filipino worker candidates (OFWCs) that had been reported from licensed laboratories to the National STD/AIDS Cooperative Central Laboratory in Manila between 2002 and 2004. FINDINGS: From over 144 000 blood-screening results, the HIV prevalence was 0.006% in BDs and 0.001% in OFWCs; that of HBV was 4.2% in both groups; and that of HCV was 0.3% in BDs and 0.9% in OFWCs. Males were at increased risk of both HBV and HCV; among OFWCs, younger women were at increased risk. Laboratories that tested sequentially but stopped testing after the first positive result were far less likely to detect HCV, indicating that sequential testing protocols may underestimate HCV and HIV prevalence. OFWCs were at low risk of HIV, and the risk of testing positive for these viruses was not increased among OFWCs applying for a repeated work visa, compared with first time-applicants. CONCLUSION: Based on these data, we conclude that HIV is rare in the Philippines. In contrast with prior reports, we found no evidence that OFWCs constitute a high-risk group for HIV. Further research is needed to understand why younger women are at increased risk of acquiring HBV.