USING KDNA MINICIRCLE SUBCLASS RELATIVE ABUNDANCE TO DIFFERENTIATE LEISHMANIA (L.) INFANTUM FROM LEISHMANIA (L.) AMAZONENSIS

1 Background Leishmaniasis is a neglected disease caused by many Leishmania species, belonging to subgenera Leishmania (Leishmania) and Leishmania (Viannia). Several qPCR-based molecular diagnostic approaches have been reported for detection and quantification of Leishmania species. Many of these approaches use the kinetoplast DNA (kDNA) minicircles as target sequence, because they contain conserved regions and are in high-copy number. However, due to sequence similarity between Leishmania species, most of the available qPCR assays had potential cross-species amplification. Previous works demonstrated discrimination between L. (Leishmania) and L. (Viannia) by SYBR green-based qPCR assays designed on kDNA, followed by melting or high resolution melt (HRM) analysis. Importantly, these approaches cannot fully distinguish L. (L.) infantum from L. (L.) amazonensis, which can coexist in the same geographic area. An accurate diagnostic method for distinction between these two species is necessary to provide prognosis, treatment and monitor disease evolution. 2 Methods DNA from 18 strains/isolates of L. (L.) infantum, L. (L.) amazonensis, L. (V.) braziliensis, L. (V.) panamensis, L. (V.) guyanensis, and 62 clinical samples from L. (L.) infantum-infected dogs were used in this study. All samples were amplified by a previously developed qPCR (qPCR-ML). Selected PCR products were directly and bidirectionally sequenced using an ABI PRISM 310 Genetic Analyzer. Sequences were manually edited and aligned in MUSCLE. Based on the obtained sequences, a new SYBR-green qPCR assay (qPCR-ama) intended to amplify a minicircle subclass more abundant in L. (L.) amazonensis was designed. 3 Results The novel qPCR-ama assay achieved a detection limit of 0.1 pg of parasite DNA and did not show cross amplification with host DNA or Trypanosoma cruzi. The qPCR-ama showed positive results also with L. (L.) infantum strains, but the Ct values were dramatically increased compared to qPCR-ML (13-19 cycles). Therefore, combined results of qPCR-ML and qPCR-ama allowed to distinguish L. (L.) infantum and L. (L.) amazonensis in 100% of tested samples, provided that template DNA was diluted so as to have Ct>25 in qPCRML. 4 Conclusions A new and affordable SYBR-green qPCR-based approach to distinguish between L. (L.) infantum and L. (L.) amazonensis was developed exploiting the major abundance of a particular minicircle sequence rather than targeting a hypothetical species-specific minicircle sequence

USING KDNA MINICIRCLE SUBCLASS RELATIVE ABUNDANCE TO DIFFERENTIATE LEISHMANIA (L.) INFANTUM FROM LEISHMANIA (L.) AMAZONENSIS

1 Background Leishmaniasis is a neglected disease caused by many Leishmania species, belonging to subgenera Leishmania (Leishmania) and Leishmania (Viannia). Several qPCR-based molecular diagnostic approaches have been reported for detection and quantification of Leishmania species. Many of these approaches use the kinetoplast DNA (kDNA) minicircles as target sequence, because they contain conserved regions and are in high-copy number. However, due to sequence similarity between Leishmania species, most of the available qPCR assays had potential cross-species amplification. Previous works demonstrated discrimination between L. (Leishmania) and L. (Viannia) by SYBR green-based qPCR assays designed on kDNA, followed by melting or high resolution melt (HRM) analysis. Importantly, these approaches cannot fully distinguish L. (L.) infantum from L. (L.) amazonensis, which can coexist in the same geographic area. An accurate diagnostic method for distinction between these two species is necessary to provide prognosis, treatment and monitor disease evolution. 2 Methods DNA from 18 strains/isolates of L. (L.) infantum, L. (L.) amazonensis, L. (V.) braziliensis, L. (V.) panamensis, L. (V.) guyanensis, and 62 clinical samples from L. (L.) infantum-infected dogs were used in this study. All samples were amplified by a previously developed qPCR (qPCR-ML). Selected PCR products were directly and bidirectionally sequenced using an ABI PRISM 310 Genetic Analyzer. Sequences were manually edited and aligned in MUSCLE. Based on the obtained sequences, a new SYBR-green qPCR assay (qPCR-ama) intended to amplify a minicircle subclass more abundant in L. (L.) amazonensis was designed. 3 Results The novel qPCR-ama assay achieved a detection limit of 0.1 pg of parasite DNA and did not show cross amplification with host DNA or Trypanosoma cruzi. The qPCR-ama showed positive results also with L. (L.) infantum strains, but the Ct values were dramatically increased compared to qPCR-ML (13-19 cycles). Therefore, combined results of qPCR-ML and qPCR-ama allowed to distinguish L. (L.) infantum and L. (L.) amazonensis in 100% of tested samples, provided that template DNA was diluted so as to have Ct>25 in qPCRML. 4 Conclusions A new and affordable SYBR-green qPCR-based approach to distinguish between L. (L.) infantum and L. (L.) amazonensis was developed exploiting the major abundance of a particular minicircle sequence rather than targeting a hypothetical species-specific minicircle sequence

Prevalence and determinants of influenza vaccination in Australians aged 40 years and over - a national survey

OBJECTIVES: To determine influenza vaccination coverage in 2001 in Australian adults aged > or = 40 years, assess awareness of and attitudes to influenza vaccine, factors associated with vaccination, and estimate uptake of free vaccine provided to those aged > or = 65 years. METHODS: National computer-assisted telephone interview (CATI) survey in October/November 2001. RESULTS: Interviews were completed with 5,266 people aged > or = 65 and 2,415 aged 40-64 years. Thirty per cent of selected households participated. Overall, 67% of respondents believed that the vaccine was somewhat to very effective in preventing influenza. Seventy-eight per cent of those aged > or = 65 years reported influenza vaccination; 89% had received it free. Independent predictors of vaccination were: belief that influenza vaccine is effective in preventing influenza (OR=13.5, 95% CI 10.6-17.2); and the presence of chronic disease (OR=1.6, 95% CI 1.3-2.0). Overall, 24% of those aged 40-64 years were vaccinated; only 34% of those who met any of the criteria for vaccination (medical risk factor, at-risk occupation, or being Aboriginal or Torres Strait Islander) reported vaccination. CONCLUSIONS: Influenza vaccine coverage was high in those aged > or = 65 years, but coverage of those at-risk aged 40-64 years remained suboptimal. Immunisation against influenza was influenced more by beliefs about the vaccine's effectiveness and existing medical risk factors, rather than socio-demographic factors such as gender and income

Additional file 2: Figure S2. of The use of kDNA minicircle subclass relative abundance to differentiate between Leishmania (L.) infantum and Leishmania (L.) amazonensis

CLUSTAL multiple alignments of 16 L. (L.) amazonensis kDNA minicircle sequences retrieved from Genbank (partial sequences). The alignment was performed by MUSCLE with default options. The boxes indicate the positions of primers LMi-amaF and LMR; the sequences perfectly matching the LMi-amaF primer are highlighted. (DOCX 16 kb