Clinico-microbiological Profile of Urinary Tract Infection with Special Reference to Uropathogenic E coli : Antibiotic susceptibility Pattern, Phylogenetic Background and Virulent Factor Distribution from West Bengal, India

Background and Objectives : Routine surveillance and monitoring studies pose a constant need to update clinicians on prevalent pathogens and rational and empirical treatment in Urinary Tract Infection (UTI). Escherichia coli (E coli) is the most commonly isolated uropathogen globally. Extended-Spectrum ?-Lactamase (ESBL) production and ?-Lactamase Inhibitor Resistance (BLIR) among these pathogens together with their uro-virulence determinants further complicate treatment approaches. This study investigated the clinico-microbiological pattern of UTI and determined the antibiotic sensitivity pattern, the phylogenetic background, and virulence determinants of E coli, the most commonly isolated uropathogen. Methods : Uropathogens isolated by urine culture from community and hospitalized patients were biochemically speciated. Antibiotic susceptibility was tested by Kirby-bauer disk diffusion method. Phylogenetic background and virulence determinants of E coli isolates were identified by PCR. SPSS 16.0 was used for statistical interpretation. Results : 45% of the urine samples showed growth positivity. 44% amongst them were E coli. All isolates were multidrug-resistant. 50% and 40% were ESBL producers and BLIR respectively. Former showed highest resistance to quinolone, fluoroquinolones, cotrimoxazole, and latter were resistant against all drugs tested except nitrofurantoin. Significant correlation existed between the ?-lactams, quinolone, fluoroquinolones, cotrimoxazole (p<0.05) resistance pattern. BLIR and ESBL E coli recorded highest prevalence of pathogenic phylogroup B2 and D respectively. Varied prevalence of fimbrial (fimH, papC, papEF, papG, GII) and toxin genes (iroN, hlyA, cnfI, i ucD, cdtBU) in ESBL, BLIR and non-ESBL isolates were observed. Their distribution was statistically significant (p=0.05). Interpretation and Conclusions : Nitrofurantoin is the drug of choice in empirical treatment of uncomplicated UTI. Aggressive and consistent investigation and health education are highly recommended for effective clinical management in UTI.

Evaluation of anti-gal enzyme-linked immunosorbent assay for the diagnosis of Indian post-kala-azar dermal leishmaniasis

Background: Elimination of kala azar from India is challenging as there are potential reservoirs of Leishmania donovani in patients with post-kala-azar dermal leishmaniasis (PKDL). The vast repertoire of carbohydrate moieties on L. donovani is known to elicit specific and strong humoral responses in patients with kala azar. Aim: The present study was undertaken to evaluate the diagnostic performances of anti-gal antibodies using enzyme-linked immunosorbent assay for successful serological diagnosis of PKDL in Indian patients and to differentiate cases of past cured visceral leishmaniasis infections. Methods: We developed Gal enzyme-linked immunosorbent assay to measure specific anti-gal IgG isotype in the sera of 71 Indian patients with PKDL. The diagnostic efficacy of the newly developed assay was evaluated for precision, sensitivity and accuracy. Results: Gal2 enzyme-linked immunosorbent assay revealed three-fold increased anti-gal titers in 71 patients with active PKDL compared to controls. Subclass enzyme-linked immunosorbent assay analysis further revealed enhanced IgG2 and IgG3 anti-gal titers in patients with PKDL compared to control subjects. The rank order for specificity and sensitivity for IgG subclasses was IgG3>IgG2>IgG4>IgG1. The area under the curve values of 0.98 and 0.99 were obtained for IgG and IgG3 Gal2 enzyme-linked immunosorbent assays respectively. Overall sensitivity and specificity were 95.7% (95% CI: 88.1–99.1) and 98.1% (95% confidence interval: 90.1–99.9), and 98.5% (95% CI: 92.4–99.9) and 98.1% (95% CI: 90.1–99.9), respectively. Intra-assay coefficient of variation was 1.5% and inter-assay coefficient of variation was 11.7%. Limitations: The Gal2 enzyme-linked immunosorbent assay needs to be further investigated in mass surveys. Conclusion: Taken together, anti-gal titers detected through Gal2 enzyme-linked immunosorbent assay can serve as an effective diagnostic tool in disease elimination setting and help in better case management in endemic districts.

Status of circulating immune complexes, IL8 titers and cryoglobulins in patients with dengue infection.

Dengue, a serious viral infection caused by the mosquito vector, Aedes aegyptii, affects about 390 million people annually from more than 125 countries across the globe. However, until now, there is no reliable clinical or laboratory indicator to accurately predict the development of dengue severity. Here, we explored critical pathophysiological determinants like IL8, circulating immune complex (CIC) and cryoglobulin in dengue-infected patients for identification of novel dengue severity biomarker(s). Totally, 100 clinically suspected dengue cases were tested by NS1 ELISA and MAC ELISA for dengue virus aetiology. For control, 49 healthy volunteers were included. Blood profiling (complete hemogram and liver function test) of patient population were done using automated cell counter and standard auto analyzer based biochemical analysis. Serum CIC was quantified by PEG precipitation. Serum cryoglobulins were estimated by Folin assay. Levels of serum IL-8 were assessed by standard sandwich ELISA kits. Patient CIC were further characterized by SDS Gel electrophoresis. Forty per cent of the cases tested positive, of which 11 patients had severe clinical manifestation. The mean ±SEM of cryoglobulin concentration for DHF, DF, and HC were 1.30±0.31, 0.59±0.08 and 0.143±0.009 μg/μl, respectively. Thus, DHF and DF patients have shown 9- and 2.2-fold increase in cryoglobulin levels; and 18- and 5-fold increased CIC, respectively compared to HC patients. The mean ±SEM of CIC-PEG index for DHF, DF and HC were 491±41.22, 146±14.19 and 27.98±2.56, respectively. Raised levels of IL8 titers were also found in all 11 DHF patients. Peak levels of CIC, cryoglobulin and IL8 titers were associated with thrombocytopenia. SDS PAGE analysis of CIC from DHF revealed the presence of at least six protein bands that were not observed in samples from DF and HC. Prediction efficacy of IL8, CIC and cryoglobulin for DHF was determined using the receiver operator characteristic curve (ROC). The area under the curve was 1.00 for IL8, 0.99 for CIC and 0.74 for cryoglobulins. Overall, the results suggest that CIC, IL-8 and cryoglobulins may serve as important laboratory parameters to monitor dengue infection progression.

Identification and glycobiological characterization of circulating immune complexes in patients with visceral leishmaniasis and post kala azar dermal leishmaniasis.

Here, we investigated the quantitative and qualitative differences in antibody classes and subclasses in serum immune complexes (ICs) of Visceral Leishmaniasis (VL), Post Kala-azar Dermal Leishmaniasis (PKDL) and different cross reactive diseases like Malaria, Leprosy, Vitiligo as compared to control subjects. IC levels were measured through a newly developed PEG ELISA, using L. donovani promastigote membrane antigen coated plate. Antibody classes and subclasses were identified using polyspecific sera and monoclonal antibodies, respectively. ICs were purified using polyethylene glycol (PEG) precipitation. Conditional logistic regression showed an association between IgG1-containing ICs and increased risk of PKDL (OR=75, P <0.05) and an association of IgG-containing ICs with VL (OR=621, P=0.001). PEG ELISA demonstrated almost 13-15 fold higher IgG containing ICs titers in VL as compared to control (P <0.001). The assay further established a significant (P <0.05) difference in the IgG containing ICs titers between VL and PKDL. The isolated ICs were further analyzed by subjecting them to one-dimensional PAGE and subsequently stained with combination of periodic acid schiff (PAS) with silver. A differential banding pattern between VL and PKDL was obtained. Four distinct bands with carbohydrate rich glycoconjugates were identified in PKDL ICs, which were absent in VL and control group. It suggests the scope for developing a novel differential diagnostic assay.

Glycobiology of Leishmania donovani.

Leishmania donovani, the causative organism of visceral leishmaniasis (VL) is one of the deadliest of the entire known Leishmania species. This protozoan parasite displays immense adaptability to survive under extremely harsh conditions. Cell surface glycoconjugates play a pivotal role in parasite virulence and infectivity. This review mainly highlights on the importance of these molecules and their reported roles with special emphasis on L. donovani sialobiology. The recently evolved information reported by our group regarding the identification and characterization of sialoglycans and their possible mode(s) of acquisition as also the detailed identification, characterization of anti-O-acetylated sialic acid (anti-OAcSA) antibodies and their emerging biological roles, notably as molecules that may aid in host defense against the pathogen has been vividly discussed in this review.