# Presentation: 142988

## A. Discovery of New Genes related to Leishmania Pathogenesisand as Biomarkers of Attenuation Using a Genomic MicroarrayB. Multiplex PCR Microarray Assay to Detect Pathogens inBlood

- Robert Duncan, PhD
- Site Visit Presentation

## Meeting the Challenge of Leishmaniasis: harvesting the benefits of the genomic era

**A method to rapidly identify virulence related genes: The Microarray**

**Goals:**
**Genetic mechanisms of *****Leishmania *****pathogenesis**
**Genetic characterization of live attenuated vaccine candidates**
**Better diagnostics based on genetic technology**
**Biomarkers of vaccine safety**

## Rationale for microarray characterization of the centrin-deleted cell line

- Urgent need for a vaccine against leishmaniasis—live, attenuated approach
- Vaccine candidate must be safe—genetically stable to avoid reversion
- Global gene expression and identified biomarkers to measure genetic stability
- Focus CBER research to make a unique scientific contribution

## L. d. Argininosuccinate synthase (22C7)

- _ __A  B  C  _

- _  _  _A  B  C  _

- _ __A  B  C  _

- _ __A  B  C  _

- _ __A  B  C  _

- _ __A  B  C  _

- Log Pro

- Log Am

- Stat Am

- KO Am

- KO AB Am

- PCD Am

- 22C7

- 24B4

- Mean
- intensity

- 125
- +/- 52

- 17
- +/- 8

- 55
- +/- 13

- 38
- +/- 8

- 19
- +/- 5

- 16
- +/- 1

- Placement on a critical metabolic pathway and reproducible pattern of expression make *L.d. *argininosuccinate synthase a potential biomarker of attenuation

## The hypothetical conserved, 27.6kD protein (46G8) L. d. homologue of LmjF28.0980

- Ld 46G8
- library clone

- _Probe_

- _ __A  B  C  _

- _  _  _A  B  C  _

- _ __A  B  C  _

- _ __A  B  C  _

- _ __A  B  C  _

- _ __A  B  C  _

- Log Pro

- Log Am

- Stat Am

- PCD Am

- KO Am

- KO AB Am

- Mean
- intensity

- 158
- +/- 17

- 31
- +/- 8

- 219
- +/- 72

- 133
- +/- 23

- 96
- +/- 38

- 1
- +/- 5

- Trypanosomatid restriction and high level of conservation suggest a critical function unique to the flagellated parasite physiology and the reproducible pattern of expression make *L.d. *p27 a potential biomarker of attenuation

## Summary: characterization of gene expression in the centrin-deleted cell line

- Differentially expressed genes identified and validated
- Selected genes with potential as biomarkers of attenuation further characterized
- Characterized genes reveal physiological correlates of centrin deletion
- Characterization of newly described gene function may lead to better understanding of *Leishmania *pathogenesis

## Transfusion blood safety has improved with pathogen testing

Increasing number of known potential infectious agents and emerging threats, including bioterrorism increases the burden of testing

Urgent need for methods to streamline and consolidate testing: nucleic acid tests (NAT), real-time PCR, microarrays, nanotechnology

Multiplex potential of a pathogen detection microarray assay

- Increasing number of known potential infectious agents and emerging threats, including bioterrorism increases the burden of testing
- Urgent need for methods to streamline and consolidate testing: nucleic acid tests (NAT), real-time PCR, microarrays, nanotechnology
- Multiplex potential of a pathogen detection microarray assay

**Meeting the Challenge of Blood Safety: harvesting the benefits of new technologies**

## Microarray for Detection of Blood-borne and BT Pathogens

_**Bacteria, and Parasites**_
- Ba: *Bacillus anthracis *(**anthrax**)
- Ft:  *Francisella tularensis *(**tularemia**)
- LT: *Leishmania* /*Trypanosoma *
- Yp: *Yersinia pestes* and *pseudotuberculosis*
**       **(**plague**)

- Yp

** ****4 internal control probes (Human rRNA gene)**

_**Blood Borne Viruses**_
- WNV: West Nile Viruses
- HCV: Hepatitis C Viruses
- HBV: Hepatitis B Viruses
- HIV: Human Immunodeficiency Viruses
- HTLV: Human T-cell Leukemia Viruses

- IC

- IC

- IC

- IC

- Ba

- Ft

_**Bioterror Viruses**_
- POX: Pox viruses
- VAC: Vaccinia
- VAR: Variola (**Smallpox**)
- MPV: Monkeypox Viruses
- CPV: Cowpox Viruses
- NOVAC: All Pox viruses but Vaccinia
- EBO: Ebola Viruses
- VE: Venezuelan Equine Enceph. Virus
- VETD: VE Trinidad Donkey
- MBG: Marburg Viruses

- IC

- LT