Puja Tiwary

Diagnosis of Indian Visceral Leishmaniasis by Nucleic Acid Detection Using PCR

Background PCR based diagnosis for Visceral Leishmaniasis (VL), despite numerous published primers, remains far from being applied in the field. The present study was planned to design a Leishmania specific diagnostic assay and to evaluate its sensitivity and specificity on a sample size, which to the best of our knowledge is the largest ever screened in one study. Methods Leishmania specific primers were developed using 18S rRNA gene and their sensitivity was evaluated on 500 parasitologically confirmed patients with VL and 25 Post Kala-azar Dermal Leishmaniasis (PKDL) patients. Specificity was calculated on 250 healthy endemic controls, 250 healthy non endemic controls and 250 non leishmanial diseases like malaria. Results Our PCR assay had a sensitivity of 87.8% (95%CI: 84.1–89.8) using 200 µL of patients peripheral-blood. Specificity was absolute in non-endemic healthy controls and in subjects with different diseases while in endemic controls it was 84% (95%CI: 78.9–88.0). Its overall specificity was 94.6% (95%CI-92.8–96.1). Conclusions The PCR assay developed is sensitive enough to detect the 18S rRNA gene in an amount equivalent to a single parasite or less in a one million human cell environment. The high sensitivity of this PCR diagnostic test with relatively non-invasive peripheral blood sampling method opens up the possibility of its deployment in field for the routine diagnosis of VL.

Seasonal Variation in the Prevalence of Sand Flies Infected with Leishmania donovani

Background Visceral Leishmaniasis (VL) is a life threatening neglected infectious disease in the Indian subcontinent, transmitted by the bite of female sand flies. Estimation of the infectivity in the vector population, collected in different seasons, may be useful to better understanding the transmission dynamics of VL as well as to plan vector control measures. Methodology We collected sand flies from highly endemic regions of Bihar state, India for one year over three seasons. The species of the sand flies were confirmed by species-specific PCR-RFLP. Leishmania donovani infection was investigated in 1397 female Phlebotomus argentipes using PCR, targeting the Leishmania specific minicircle of the kDNA region. Further, the parasitic load in the infected sand flies was measured using quantitative PCR. Conclusion Though sand flies were most abundant in the rainy season, the highest rate of infection was detected in the winter season with 2.84% sand flies infected followed by the summer and rainy seasons respectively. This study can help in vector elimination programmes and to reduce disease transmission.

Genetic and functional evaluation of the role of CXCR1 and CXCR2 in susceptibility to visceral leishmaniasis in north-east India

BackgroundIL8RA and IL8RB, encoded by CXCR1 and CXCR2, are receptors for interleukin (IL)-8 and other CXC chemokines involved in chemotaxis and activation of polymorphonuclear neutrophils (PMN). Variants at CXCR1 and CXCR2 have been associated with susceptibility to cutaneous and mucocutaneous leishmaniasis in Brazil. Here we investigate the role of CXCR1/CXCR2 in visceral leishmaniasis (VL) in India.MethodsThree single nucleotide polymorphisms (SNPs) (rs4674259, rs2234671, rs3138060) that tag linkage disequilibrium blocks across CXCR1/CXCR2 were genotyped in primary family-based (313 cases; 176 nuclear families; 836 individuals) and replication (941 cases; 992 controls) samples. Family- and population-based analyses were performed to look for association between CXCR1/CXCR2 variants and VL. Quantitative RT/PCR was used to compare CXCR1/CXCR2 expression in mRNA from paired splenic aspirates taken before and after treatment from 19 VL patients.ResultsFamily-based analysis using FBAT showed association between VL and SNPs CXCR1_rs2234671 (Z-score = 2.935, P = 0.003) and CXCR1_rs3138060 (Z-score = 2.22, P = 0.026), but not with CXCR2_rs4674259. Logistic regression analysis of the case-control data under an additive model of inheritance showed association between VL and SNPs CXCR2_rs4674259 (OR = 1.15, 95%CI = 1.01-1.31, P = 0.027) and CXCR1_rs3138060 (OR = 1.25, 95%CI = 1.02-1.53, P = 0.028), but not with CXCR1_rs2234671. The 3-locus haplotype T_G_C across these SNPs was shown to be the risk haplotype in both family- (TRANSMIT; P = 0.014) and population- (OR = 1.16, P = 0.028) samples (combined P = 0.002). CXCR2, but not CXCR1, expression was down regulated in pre-treatment compared to post-treatment splenic aspirates (P = 0.021).ConclusionsThis well-powered primary and replication genetic study, together with functional analysis of gene expression, implicate CXCR2 in determining outcome of VL in India.

Prevalence of Sand Flies and Leishmania donovani Infection in a Natural Population of Female Phlebotomus argentipes in Bihar State, India

Leishmaniasis is a vector-borne disease, and in the Indian subcontinent the female Phlebotomus argentipes is the vector for Leishmania donovani. However, data on the extent of sand fly infection rates in natural settings using molecular methods have not been extensively reported in India. In this study a PCR technique was applied targeting the 18S rRNA encoding region to determine the prevalence of Leishmania infection in female P. argentipes captured in the field. For this study, sand flies were collected from 897 houses selected from 50 villages endemic for visceral leishmaniasis (VL) in Muzaffarpur district, Bihar state, using CDC miniature light traps and mouth aspirators. A total of 14,585 sand flies were collected of which 449 were female P. argentipes divided into 132 pools. Molecular detection using PCR targeting the 18S rRNA gene was carried out for the identification of P. argentipes and Leishmania. The overall prevalence of infection was 4.90-17.37% for L. donovani in female P. argentipes in endemic regions of Bihar state. In this study no correlation was found between the presence of infected sand flies and the occurrence of clinical VL. This study provides the first report evaluating the prevalence of Leishmania infection in sand flies in a region endemic for VL in India. Sergentomyia species are the most common species of sand fly. Knowledge of the infection rate in female P. argentipes may help in predicting severity of disease and in vector elimination programs.

PCR-RFLP based method for molecular differentiation of sand fly species Phlebotomus argentipes, Phlebotomus papatasi, and Sergentomyia babu found in India.

ABSTRACT PCR-Restriction fragment length polymorphism is a time saving and accurate technique to differentiate closely related organisms. In the regions endemic for visceral leishmaniasis in India, various species of morphological similar sand fly exist but only female Phlebotomus argentipes is the vector for visceral leishmaniasis. In the current study primers were designed targeting the 18S rRNA encoding gene that showed amplification in all the major sand fly species found in India. The amplified fragments were further digested using the HinfI or HpaII restriction enzymes. Each of the restriction enzyme produced a species specific restriction patterns, which can easily be used to identify specific sand fly species. This technique can be used in the identification of sand fly species.

Comparative Evaluation of Blood and Serum Samples in Rapid Immunochromatographic Tests for Visceral Leishmaniasis

ABSTRACT Rapid diagnostic tests (RDTs) based on the detection of specific antibodies in serum are commonly used for the diagnosis of visceral leishmaniasis (VL). Several commercial kits are available, and some of them allow the use of whole-blood samples instead of serum. An RDT is much more user-friendly for blood samples than for serum samples. In this study, we examined the sensitivities and specificities of six different commercially available immunochromatographic tests for their accuracy in detecting Leishmania infection in whole blood and serum of parasitologically confirmed VL cases. This study was performed in areas of India and Nepal where VL is endemic. A total of 177 confirmed VL cases, 208 healthy controls from areas of endemicity (EHCs), 26 malaria patients (MP), and 37 tuberculosis (TB) patients were enrolled. The reproducibilities of the blood and serum results and between-reader and between-laboratory results were tested. In India, the sensitivities of all the RDTs ranged between 94.7 and 100.0%, with no significant differences between whole blood and serum. The specificities ranged between 92.4 and 100.0%, except for the specificity of the Onsite Leishmania Ab RevB kit, which was lower (33.6 to 42.0%). No differences in specificities were observed for blood and serum. In Nepal, the sensitivities of all the test kits, for whole-blood as well as serum samples, ranged between 96.3 and 100.0%, and the specificities ranged between 90.1 and 96.1%, again with the exception of that of the Onsite Leishmania Ab RevB test, which was markedly lower (48.7 to 49.3%). The diagnostic accuracies of all the tests, except for one brand, were excellent for the whole-blood and serum samples. We conclude that whole blood is an adequate alternative for serum in RDTs for VL, with sensitivities and specificities comparable to those obtained in serum samples, provided that the test kit is of overall good quality.

Establishing, Expanding, and Certifying a Closed Colony of Phlebotomus argentipes (Diptera: Psychodidae) for Xenodiagnostic Studies at the Kala Azar Medical Research Center, Muzaffarpur, Bihar, India

Abstract This pilot project was preliminary and essential to a larger effort to define the ability of certain human-subject groups across the infection spectrum to serve as reservoirs of Leishmania donovani infection to sand flies in areas of anthroponotic transmission such as in Bihar state, India. This is possible only via xenodiagnosis of well-defined subject groups using live vector sand flies. The objective was to establish at the Kala Azar Medical Research Center (KAMRC), Muzaffarpur, Bihar, India, a self-sustaining colony of Phlebotomus argentipes (Annandale & Brunneti), closed to infusion with wild-caught material and certified safe for human xenodiagnosis. Prior to this endeavor, no laboratory colony of this vector existed in India meeting the stringent biosafety requirements of this human-use study. From March through mid-December, 2015, over 68,000 sand flies were collected in human dwellings and cattle sheds using CDC-type light traps over 254 nights. Blood-fed and gravid P. argentipes females were selected and placed individually in isoline-rearing vials for oviposition, and >2,500 egg clutches were harvested. Progeny were reared according to standard methods, providing a continuous critical mass of F1 males and females to stimulate social feeding behavior. With construction of a large feeding cage and use of a custom-made rabbit restrainer, the desired level of blood-feeding on restrained rabbits was achieved to make the colony self-sustaining and expand it to working level. Once self-sustaining, the colony was closed to infusion with wild-caught material and certified free of specific human pathogens.

Association of Interleukin-18 Gene Polymorphism with Susceptibility to Visceral Leishmaniasis in Endemic Area of Bihar, an Indian Population

Interleukin-18 (IL-18) is a cytokine that mediates Th1 response by inducing interferon-gamma (IFN-γ) production in T cells and natural killer cells. Genetic polymorphisms in the IL-18 gene have been found to be associated with its expression in cancer, tuberculosis, HBV infection, and various other diseases. Lower plasma level of IL-18 in visceral leishmaniasis (VL) patients might be associated with polymorphisms in the regulating or coding region of the gene. Three single nucleotide polymorphisms (SNPs), rs1946519 (−656 G/T) and rs187238 (−137 G/C) in the promoter region and rs549908 (+105 A/C) in the codon region, were genotyped in 204 parasitological confirmed VL patients and 267 controls with no past history of VL. For each locus, polymerase chain reaction (PCR) followed by restriction digestion was performed. IL-18 expression in peripheral blood mononuclear cells (PBMC) collected from VL patients and controls was measured by quantitative real-time RT-PCR. Distribution of G allele at position −656 (P < 0.0001) and double haplotypes GGC/GGA (P = 0.05) were found to be significantly associated with controls while genotypes TT (P < 0.0001) and single haplotypes TGA (P = 0.0002), with cases. The inheritance of G allele at the position −656 might be considered as a protective allele for VL.

Cloning, expression and purification of L. Donovani specific antigen for serodiagnosis of visceral leishmaniasis

Background Rapid diagnostic test using rk39 antigen is widely used for visceral leishmaniasis. However it detects anti-rk39 antibodies in 20-32% of endemic healthy individuals. In search for a better biomarker of infection, we identified a protein of molecular weight 70 kDa (BHUP1), specifically recognized by sera of visceral leishmaniasis (VL) patients. Methods The protein was cloned as His-tagged fusion protein and purified. We evaluated the sensitivity and specificity of this protein in an enzyme linked immunosorbant assay (ELISA) format in comparison to the rk39 antigen using sera collected from various groups of individuals. Results The sensitivity of rBHUP1 was 96.5% compared to 98.8% with rk39. For healthy controls from non endemic and endemic regions, the specificity of rBHUP1 was 100% and 95.6% compared to 100% and 84.9% for rk39, respectively. For other infectious diseases such as malaria, tuberculosis, viral fever, etc., specificity of rBHUP1 was as low as 74.5% when compared to 94% of rk39. At six month and one year follow-up, 74% and 22.5% patients tested positive with rBHUP1, respectively, compared to 97% and 77.4% with rk39 antigen. Conclusion Though the high sensitivity and specificity of rBHUP1 antigen for VL and healthy controls would have made it a good diagnostic biomarkers, however, its non-specific reaction with other infectious diseases limit its utility.

Identification and Functional Validation of a Biomarker for the Diagnosis of Miltefosine Relapse during Visceral Leishmaniasis

Miltefosine is the only orally administrable drug for the treatment of leishmaniasis. But in recent years, a decline in its efficacy points toward the emergence of resistance to this drug. Knowledge of biomarkers for miltefosine resistance may be beneficial for proper selection of treatment regimen. Splenic aspirates were collected and parasites cultured from patients relapsed after initial cure (N = 15) and successfully treated (N = 15) with miltefosine. Differential expression of genes in miltefosine-resistant strains was examined by DNA microarray and validated by real-time reverse transcription polymerase chain reaction and Western blotting. Of 669 upregulated genes, the cysteine protease-like protein of calpain family (GenBank: CBZ34784) was found to be significantly overexpressed in resistant parasite strains and only anti-calpain antibodies showed its presence in the sera of relapse patients through Western blotting. Calpain family cysteine protease-like protein can be useful as a potential biomarker of miltefosine unresponsiveness.