Genotypic resistance profiles in antiretroviral-naive HIV-1 infections before and after initiation of first-line HAART: impact of polymorphism on resistance to therapy.

Genotypic testing using TRUGENE was performed for treatment-naive, human immunodeficiency virus type 1 (HIV-1)-infected patients at baseline and after initiation of protease inhibitor (PI)-based highly active antiretroviral therapy (HAART) regimens. The genetic diversity of HIV-1 pol sequences from 92 CRF01_AE and 21 B strains was compared. Subsequently, the impact of polymorphism on resistance to therapy was studied in CRF01_AE-infected (n=29) and subtype B-infected (n=14) patients. At baseline, the differences between CRF01_AE and B strain were mainly observed in the minor mutations L10I/V, M36I and L63P/I/H (P<0.001, chi(2)). In the reverse transcriptase sequence, M41L and T215Y/S were more common in patients infected with subtype B virus (P<0.05, chi(2)). Although all patients treated with PI-based HAART had pre-existing minor mutations, a low prevalence of resistance to PIs was observed (5/43; 11.6%). Moreover, major mutations (D30N and N88D) conferring resistance to PIs were found in patients infected with subtype B strain. In conclusion, polymorphisms at the protease region may not reduce PI susceptibility during treatment. However, this study also revealed the difference in natural mutations among subtypes, which may affect the manifestation of drug resistance.

Development and evaluation of an immunochromatographic assay to detect serum anti-leptospiral lipopolysaccharide IgM in acute leptospirosis.

Leptospirosis is a common life-threatening disease worldwide. However, its diagnosis is frequently ineffective because the gold standard bacterial culture and microscopic agglutination test (MAT) are usually positive 1-2 weeks after the disease onset. We thus developed an immunochromatographic assay (LEPkit) to detect serum anti-leptospiral lipopolysaccharide (LPS) IgM for rapid diagnosis of acute leptospirosis. Using referenced sera of 77 leptospirosis and 91 non-leptospirosis cases, LEPkit yielded 97.4% sensitivity, 94.5% specificity, 93.8 positive predictive value (PPV), 97.7% negative predictive value (NPV), and 95.8% accuracy. The stability of this kit stored for up to 18 months and its reproducibility were confirmed. Testing in 74 new cases using samples at admission-phase and subsequent paired samples (total n = 135), overall sensitivity was 98.5%, whereas that of culture and single MAT (≥1:400) was 15.6% and 35.6%, respectively. When only the samples at admission-phase were used (n = 74), the sensitivity remained at 98.7%, whereas that of culture and single MAT (≥1:400) was 28.4% and 13.5%, respectively. In summary, our LEPkit was far more effective than any conventional methods for the diagnosis of acute leptospirosis, especially within the first few days after the disease onset. The ease of use, stability and reproducibility further enhance its feasibility for clinical use on-site.

Early diagnosis of leptospirosis by immunoglobulin M immunoblot testing.

There is an urgent need for the development of serodiagnostic approaches with improved sensitivity for patients with acute leptospirosis. Immunoblots were performed on 188 sera collected from 74 patients with laboratory-confirmed early leptospiral infection to detect immunoglobulin M (IgM) antibodies to antigens pooled from 10 leptospiral strains prevalent in Thailand. Sera from patients with other febrile diseases served as controls. IgM reactivity to seven distinct antigens, with apparent molecular masses of 14 to 18, 19 to 23, 24 to 30, 32, 35/36, 37, and 41/42 kDa, was observed. The low-molecular-mass 14- to 18-kDa band was the most frequently detected antigen, being recognized in sera from 82.4% of patients during the first 3 days after the onset of symptoms. We evaluated the accuracy of the IgM immunoblot (IgM-IB) test by using reactivity to the 14- to 18-kDa band and/or at least two bands among the 19- to 23-, 24- to 30-, 32-, 35/36-, 37-, and 41/42-kDa antigens as the diagnostic criterion. The sensitivities of the IgM-IB test and the microscopic agglutination test (MAT) were 88.2% and 2.0%, respectively, with sera from patients 1 to 3 days after the onset of symptoms. In contrast, the IgM-IB test was positive with only 2/48 (4.2%) sera from patients with other febrile illnesses. The high sensitivity and specificity of the IgM-IB test for acute leptospirosis would provide greatly improved diagnostic accuracy for identification of patients who would benefit from early antibiotic intervention. In addition, the antigens identified by the IgM-IB test may serve as components of a rapid, accurate, point-of-care diagnostic test for early leptospirosis.