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author:("tiwari, pufa")
1.  PCR-RFLP based method for molecular differentiation of sand fly species Phlebotomus argentipes, Phlebotomus papatasi and Sergentomyia babu found in India 
Journal of medical entomology  2012;49(6):1515-1518.
PCR- Restriction fragment length polymorphism (RFLP) is a time saving and accurate technique to differentiate closely related organisms. In the regions endemic for visceral leishmaniasis (VL) in India, various species of morphological similar sand fly exists but only female Phlebotomus argentipes is the vector for VL. In the present 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 Hinf I or Hpa II restriction enzymes. Each of the restriction enzyme produced species specific restriction patterns, which can easily be used to identify specific sand fly species. This technique can be employed in the identification of the species of the sand flies.
PMCID: PMC3533248  PMID: 23270185
18S rRNA; Leishmaniasis; PCR; Phlebotomine; RFLP; Sand fly
2.  Genetic and functional evaluation of the role of DLL1 in susceptibility to visceral leishmaniasis in India 
Chromosome 6q26–27 is linked to susceptibility to visceral leishmaniasis (VL) in Brazil and Sudan. DLL1 encoding the Delta-like 1 ligand for Notch 3 was implicated as the etiological gene. DLL1 belongs to the family of Notch ligands known to selectively drive antigen-specific CD4 T helper 1 cell responses, which are important in protective immune response in leishmaniasis. Here we provide further genetic and functional evidence that supports a role for DLL1 in a well-powered population-based study centred in the largest global focus of VL in India. Twenty-one single nucleotide polymorphisms (SNPs) at PHF10/C6orf70/DLL1/FAM120B/PSMB1/TBP were genotyped in 941 cases and 992 controls. Logistic regression analysis under an additive model showed association between VL and variants at DLL1 and FAM120B, with top associations (rs9460106, OR=1.17, 95%CI 1.01–1.35, P=0.033; rs2103816, OR=1.16, 95%CI 1.01–1.34, P=0.039) robust to analysis using caste as a covariate to take account of population substructure. Haplotype analysis taking population substructure into account identified a common 2-SNP risk haplotype (frequency 0.43; P=0.028) at FAM120B, while the most significant protective haplotype (frequency 0.18; P=0.007) was a 5-SNP haplotype across the interval 5’ of both DLL1 (negative strand) and FAM120B (positive strand) and extending to intron 4 of DLL1. Quantitative RT/PCR was used to compare expression of 6q27 genes in paired pre- and post-treatment splenic aspirates from VL patients (N=19). DLL1 was the only gene to show differential expression that was higher (P<0.0001) in pre- compared to post-treatment samples, suggesting that regulation of gene expression was important in disease pathogenesis. This well-powered genetic and functional study in an Indian population provides evidence supporting DLL1 as the etiological gene contributing to susceptibility to VL at Chromosome 6q27, confirming the potential for polymorphism at DLL1 to act as a genetic risk factor across the epidemiological divides of geography and parasite species.
doi:10.1016/j.meegid.2012.04.017
PMCID: PMC3651914  PMID: 22561395
visceral leishmaniasis; DLL1; genetic association; Notch signalling
3.  Prevalence of Sand Flies and Leishmania donovani Infection in a Natural Population of Female Phlebotomus argentipes in Bihar State, India 
Abstract
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.
doi:10.1089/vbz.2011.0808
PMCID: PMC3366094  PMID: 22217179
Leishmania donovani; Phlebotomus argentipes; 18S rRNA; Gene; PCR; Sand fly; Visceral leishmaniasis
4.  Seasonal Variation in the Prevalence of Sand Flies Infected with Leishmania donovani 
PLoS ONE  2013;8(4):e61370.
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.
doi:10.1371/journal.pone.0061370
PMCID: PMC3621828  PMID: 23585896
5.  Genetic and functional evaluation of the role of CXCR1 and CXCR2 in susceptibility to visceral leishmaniasis in north-east India 
BMC Medical Genetics  2011;12:162.
Background
IL8RA 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.
Methods
Three 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.
Results
Family-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).
Conclusions
This well-powered primary and replication genetic study, together with functional analysis of gene expression, implicate CXCR2 in determining outcome of VL in India.
doi:10.1186/1471-2350-12-162
PMCID: PMC3260103  PMID: 22171941
6.  No evidence for association between SLC11A1 and visceral leishmaniasis in India 
BMC Medical Genetics  2011;12:71.
Background
SLC11A1 has pleiotropic effects on macrophage function and remains a strong candidate for infectious disease susceptibility. 5' and/or 3' polymorphisms have been associated with tuberculosis, leprosy, and visceral leishmaniasis (VL). Most studies undertaken to date were under-powered, and none has been replicated within a population. Association with tuberculosis has replicated variably across populations. Here we investigate SLC11A1 and VL in India.
Methods
Nine polymorphisms (rs34448891, rs7573065, rs2276631, rs3731865, rs17221959, rs2279015, rs17235409, rs17235416, rs17229009) that tag linkage disequilibrium blocks across SLC11A1 were genotyped in primary family-based (313 cases; 176 families) and replication (941 cases; 992 controls) samples. Family- and population-based analyses were performed to look for association between SLC11A1 variants and VL. Quantitative RT/PCR was used to compare SLC11A1 expression in mRNA from paired splenic aspirates taken before and after treatment from 24 VL patients carrying different genotypes at the functional promoter GTn polymorphism (rs34448891).
Results
No associations were observed between VL and polymorphisms at SLC11A1 that were either robust to correction for multiple testing or replicated across primary and replication samples. No differences in expression of SLC11A1 were observed when comparing pre- and post-treatment samples, or between individuals carrying different genotypes at the GTn repeat.
Conclusions
This is the first well-powered study of SLC11A1 as a candidate for VL, which we conclude does not have a major role in regulating VL susceptibility in India.
doi:10.1186/1471-2350-12-71
PMCID: PMC3128845  PMID: 21599885
SLC11A1; visceral leishmaniasis; genetic susceptibility
7.  Diagnosis of Indian Visceral Leishmaniasis by Nucleic Acid Detection Using PCR 
PLoS ONE  2011;6(4):e19304.
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 patient's 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.
doi:10.1371/journal.pone.0019304
PMCID: PMC3084819  PMID: 21559398

Results 1-7 (7)