Final Report Summary - TM-REST (A new platform for fast molecular detection of MDR and XDR resistant strains of m. tuberculosis and of drug resistant malaria)
The increasing threat of infections due to m. tuberculosis (TB) including multidrug resistant tuberculosis (MDR-TB) and extensively drug-resistant (XDR) m. tuberculosis infections, poses important questions that call for the development of integrated tools for rapid diagnosis. In the specific case of TB, an integrated rapid diagnostic approach should be able to permit species identification, drug susceptibility testing (DST) and molecular typing.
Another poverty related disease, malaria, caused by the protozoan parasite plasmodium falciparum, kills between 1.5 million and 3 million people each year. Deaths from malaria are increasing, especially in Africa, largely because of antimalarial drug resistance. A rapid diagnostic tool for drug resistance would be affordable within European healthcare systems and advantageous in countries where patients are treated in walk-in clinics with little follow-up.
With the aim to release an highly automated commercial product, namely a diagnostic kit for TB and malaria, with minimum hands-on time and based on the same technology, we developed, tested and validated new rapid diagnostic tests for molecular diagnosis and monitoring of TB and its drug resistant variants, as well as for the detection of malaria using a lab-on-chip (LoC) platform.
The in-checkTM technology was based on an integrated polymerase chain reaction (PCR) and a deoxyribonucleic acid (DNA) microarray for the end point analysis and consisted in a single disposable device, or biochip, and on associated specific instruments. The main advancement over existing technology consisted in the possibility to perform PCR and hybridisation in a single device at competitive costs, using a higher number of genetic probes by integrating multiple PCR chambers and low density array with faster and more stable amplification and hybridisation reactions through optimised and controlled thermal ramps and profile.
The developed test could identify reliably, both from clinical samples and strains, and on the same chip m. tuberculosis complex, main non-tuberculous species and the most frequent mutations leading to the MDR phenotype with high sensitivity and specificity.
The malaria assay allowed the specific identification of all human plasmodium parasite species. In addition the LoC could reliably detect drug resistant parasites carrying the acknowledged resistance mutations in Pfcrt (chloroquine resistance), Pfdhfr (pyrimethamine resistance) and Pfcytb (atovaquone resistance).
The micro fluidic technology was constantly improved with the aim to release an highly automated product with minimum hands-on time and a sample extraction protocol with very low level of biosafety requirements. The platform was already commercially available for the detection and typing of human strains of onfluenza A and B viruses, including the avian flu strain H5N1. With the development of the chips for TB and malaria, this technology could be widely adopted, especially in TB and malaria endemic countries where it could have significant impact on morbidity and mortality.
The main objective of the TM-REST project were to:
1. develop, test and validate a specific diagnostic assay on a LoC based new platform (In-checkTM) for the molecular diagnosis and monitoring of TB and its DR variants and for the support and guidance of therapeutic interventions. This tool would allow the identification of DR-TB by the use of selected genomic markers. The integrated PCR and microarray LoC and the solid state microarray optical reading tool represented a clear innovation over the conventional readers for their robustness, simplicity of use and low cost. Non-invasive and quantitative methods would be implemented, including quality assurance (QA) and biosafety aspects.
2. develop, test and validate a specific diagnostic assay able to detect specific markers of drug resistant variants of malaria, another poverty related disease using the same platform technology and methodology.
The In-CheckTM technology was based on an integrated PCR and a DNA microarray for the endpoint analysis and consisted in a single disposable device, biochip, and on associated specific instruments.
The LoC provided an all in one device for fast PCR amplification and detection of targets on a low density microarray by integrating all the functions needed to identify multiple oligonucleotide sequences in a sample, namely an integrated high speed PCR reactor, a low density microarray and microfluidic handling.
The LoC was composed of two modules:
1. PCR module, thermally driven by the In-CheckTM temperature control system (TCS)
2. microarray module, used to detect the amplified labelled DNA sequences through hybridisation on a low density microarray.
The dedicated low cost set of optimised instruments consisted of
1. a compact TCS for amplification and hybridisation
2. a compact, solid state, user friendly optical reader able to analyse the microarray automatically and to provide an easy to read diagnostic report within few seconds
3. a personal computer (PC).
The most important phase in building up a customised chip consisted on the microarray lay out definition. This phase, performed by means of a 'design rule manual' developed by ST Microelectronics, allowed the construction of a microarray on which each position was well known. The microarray was designed in order to have two different sub-microarrays in the same chip with a plane of symmetry, which allowed a better control in terms of microarray efficacy. In addition, the probes were printed in duplicate, one in each sub-microarray. This eliminated the risk of surface effects on the chip or non uniform washing of the microarray. The outcome of this process was a grid defining all the positions to be used by the software or the operator for the interpretation of the results.
The In-CheckTM platform integrated the advantages provided by a thermal cycler (RT and PCR) with the detection of target sequences on low density microarray. Main advancement over existing technology consisted in the possibility to perform PCR and hybridisation in a single device at competitive costs, using a higher number of genetic probes by integrating multiple PCR chambers and low density array with faster and more stable amplification and hybridisation reactions through optimised and controlled thermal ramps and profile. Additionally, the use of this platform did not require advanced molecular biology background. Training of laboratory technicians could be successfully performed in one to two days.
TB and malaria assays relied on the amplification of target genes by PCR performed on DNA extracted from both cultures and clinical samples. An additional chip based on the spoligotype technology was also designed and validated on clinical samples.
For TB, the performance of the platform was assessed using DNA extracted from both isolates and clinical specimens. Results were analysed by the signal to background ratio calculated by the array software analysis. Provisional thresholds for probe evaluation were set up performing a receiver operating characteristics (ROC) curve based on data from probes. Median signals with interquartile range for each target region were defined and thresholds were set between lowest interquartile range and highest interquartile range signals.
Selected probes identified the mycobacterium tuberculosis complex (MTBC), as well as 10 clinically relevant non-tubercular mycobacterial species. Concerning MDR-TB detection, the assay detected mutations D516V, S531L for rpoB, S315T for katG and c-15t, t-8c, t-8a for inhA. Other mutations were identified by a negative signal from wild type probes.
Regarding malaria, the LoC was tested with in vitro grown p. falciparum, single species infections of the other non-culturable parasites from experimental infections in monkeys or naturally occurring single-species infections in humans and patient samples containing mixtures of parasite species. In all cases, it allowed correct identification of all human malaria parasite species.
Results were analysed by the signal to background ratio calculated by the array software analysis. The signal to background ratio for probes 'expected on' was always very significantly higher than for probes 'expected off', although the difference varied depending on the parasitaemia in the sample. To overcome this difficulty, ratios were expressed relative to the PCR control probe and analyses were performed using this methodology.
For drug resistance, the assay was able to detect the key mutation in Pfcrt (K76T) necessary for resistance to chloroquine, as well as the initial key mutation (PfhdhfrS108N) responsible for resistance to antifolate drugs such as pyrimethamine), the additional mutation conferring increased resistance (PfdhfrC59R) and the mutation conferring very high level resistance to these drugs (PfdhfrI164L). The assay gave variable results for the detection of a key mutation for resistance to proguanil (PfdhfrA16S/V) and for detection of mutations at another codon responsible for intermediate resistance to pyrimethamine (PfdhfrI51N). Finally the LoC assay was able to detect the wild type allele of Pfcytb268Y where mutations (Pfcytb268S/C) caused resistance to atovaquone.
This integrated PCR microarray LoC represented an innovation for its ease of use and cost effectiveness. The developed platform was the first semi-automated platform able to perform the diagnosis of the two major poverty related diseases and was easily adaptable for additional diagnostics purposes. The main features of platform made it suitable for the use in reference and regional laboratories also in low and middle income countries with high burden of tuberculosis and malaria.
The developed test could reliably identify, both from clinical samples and strains and on the same chip TB complex, main non TB species and the most frequent mutations leading to the MDR phenotype with high sensitivity and specificity. A chip for the XDR phenotype was also developed but not fully validated. Less frequent mutations (L533P, H526D, S315T variation two) were identified by the absence of hybridisation with the wild type probes.
The malaria assay allowed the specific identification of all human plasmodium parasite species. In addition the LoC could reliably detect drug resistant parasites carrying the acknowledged resistance mutations in Pfcrt, Pfdhfr and Pfcytb.
The proposed technology could be widely adopted, especially in TB and malaria endemic countries where it could have significant impact on morbidity and mortality of these diseases.
The scientific work conducted under this project led to several publications in international scientific journals, such as 'Antimicrobial agents and chemotherapy' and the 'Journal of clinical microbiology' by the American Society for Microbiology (ASM), the 'Journal of antimicrobial chemotherapy' by Oxford University Press, the 'BMC infectious diseases' and the 'International journal of antimicrobial agents'. During project implementation period, results of the scientific work were also presented during scientific congresses at national, European and international levels, in particular during national conferences and meetings and the annual congresses of the ASM and of the European Society of Mycobacteriology, that were held in several countries in Europe and in the United States of America. The project website was also used and constantly updated to disseminate main findings and project achievements.
A project website at 'http://www.tm-rest.org' was developed at the beginning of the project and was composed of a public section and a restricted area, accessible by project partners only for reporting relevant project information. The website was regularly maintained and updated with most relevant progress on research activities for dissemination to the public. A database of mutations conferring resistance to antimycobacterial first and second line drugs developed by P2-FZB with contribution from project partners was also posted in the website.
Another poverty related disease, malaria, caused by the protozoan parasite plasmodium falciparum, kills between 1.5 million and 3 million people each year. Deaths from malaria are increasing, especially in Africa, largely because of antimalarial drug resistance. A rapid diagnostic tool for drug resistance would be affordable within European healthcare systems and advantageous in countries where patients are treated in walk-in clinics with little follow-up.
With the aim to release an highly automated commercial product, namely a diagnostic kit for TB and malaria, with minimum hands-on time and based on the same technology, we developed, tested and validated new rapid diagnostic tests for molecular diagnosis and monitoring of TB and its drug resistant variants, as well as for the detection of malaria using a lab-on-chip (LoC) platform.
The in-checkTM technology was based on an integrated polymerase chain reaction (PCR) and a deoxyribonucleic acid (DNA) microarray for the end point analysis and consisted in a single disposable device, or biochip, and on associated specific instruments. The main advancement over existing technology consisted in the possibility to perform PCR and hybridisation in a single device at competitive costs, using a higher number of genetic probes by integrating multiple PCR chambers and low density array with faster and more stable amplification and hybridisation reactions through optimised and controlled thermal ramps and profile.
The developed test could identify reliably, both from clinical samples and strains, and on the same chip m. tuberculosis complex, main non-tuberculous species and the most frequent mutations leading to the MDR phenotype with high sensitivity and specificity.
The malaria assay allowed the specific identification of all human plasmodium parasite species. In addition the LoC could reliably detect drug resistant parasites carrying the acknowledged resistance mutations in Pfcrt (chloroquine resistance), Pfdhfr (pyrimethamine resistance) and Pfcytb (atovaquone resistance).
The micro fluidic technology was constantly improved with the aim to release an highly automated product with minimum hands-on time and a sample extraction protocol with very low level of biosafety requirements. The platform was already commercially available for the detection and typing of human strains of onfluenza A and B viruses, including the avian flu strain H5N1. With the development of the chips for TB and malaria, this technology could be widely adopted, especially in TB and malaria endemic countries where it could have significant impact on morbidity and mortality.
The main objective of the TM-REST project were to:
1. develop, test and validate a specific diagnostic assay on a LoC based new platform (In-checkTM) for the molecular diagnosis and monitoring of TB and its DR variants and for the support and guidance of therapeutic interventions. This tool would allow the identification of DR-TB by the use of selected genomic markers. The integrated PCR and microarray LoC and the solid state microarray optical reading tool represented a clear innovation over the conventional readers for their robustness, simplicity of use and low cost. Non-invasive and quantitative methods would be implemented, including quality assurance (QA) and biosafety aspects.
2. develop, test and validate a specific diagnostic assay able to detect specific markers of drug resistant variants of malaria, another poverty related disease using the same platform technology and methodology.
The In-CheckTM technology was based on an integrated PCR and a DNA microarray for the endpoint analysis and consisted in a single disposable device, biochip, and on associated specific instruments.
The LoC provided an all in one device for fast PCR amplification and detection of targets on a low density microarray by integrating all the functions needed to identify multiple oligonucleotide sequences in a sample, namely an integrated high speed PCR reactor, a low density microarray and microfluidic handling.
The LoC was composed of two modules:
1. PCR module, thermally driven by the In-CheckTM temperature control system (TCS)
2. microarray module, used to detect the amplified labelled DNA sequences through hybridisation on a low density microarray.
The dedicated low cost set of optimised instruments consisted of
1. a compact TCS for amplification and hybridisation
2. a compact, solid state, user friendly optical reader able to analyse the microarray automatically and to provide an easy to read diagnostic report within few seconds
3. a personal computer (PC).
The most important phase in building up a customised chip consisted on the microarray lay out definition. This phase, performed by means of a 'design rule manual' developed by ST Microelectronics, allowed the construction of a microarray on which each position was well known. The microarray was designed in order to have two different sub-microarrays in the same chip with a plane of symmetry, which allowed a better control in terms of microarray efficacy. In addition, the probes were printed in duplicate, one in each sub-microarray. This eliminated the risk of surface effects on the chip or non uniform washing of the microarray. The outcome of this process was a grid defining all the positions to be used by the software or the operator for the interpretation of the results.
The In-CheckTM platform integrated the advantages provided by a thermal cycler (RT and PCR) with the detection of target sequences on low density microarray. Main advancement over existing technology consisted in the possibility to perform PCR and hybridisation in a single device at competitive costs, using a higher number of genetic probes by integrating multiple PCR chambers and low density array with faster and more stable amplification and hybridisation reactions through optimised and controlled thermal ramps and profile. Additionally, the use of this platform did not require advanced molecular biology background. Training of laboratory technicians could be successfully performed in one to two days.
TB and malaria assays relied on the amplification of target genes by PCR performed on DNA extracted from both cultures and clinical samples. An additional chip based on the spoligotype technology was also designed and validated on clinical samples.
For TB, the performance of the platform was assessed using DNA extracted from both isolates and clinical specimens. Results were analysed by the signal to background ratio calculated by the array software analysis. Provisional thresholds for probe evaluation were set up performing a receiver operating characteristics (ROC) curve based on data from probes. Median signals with interquartile range for each target region were defined and thresholds were set between lowest interquartile range and highest interquartile range signals.
Selected probes identified the mycobacterium tuberculosis complex (MTBC), as well as 10 clinically relevant non-tubercular mycobacterial species. Concerning MDR-TB detection, the assay detected mutations D516V, S531L for rpoB, S315T for katG and c-15t, t-8c, t-8a for inhA. Other mutations were identified by a negative signal from wild type probes.
Regarding malaria, the LoC was tested with in vitro grown p. falciparum, single species infections of the other non-culturable parasites from experimental infections in monkeys or naturally occurring single-species infections in humans and patient samples containing mixtures of parasite species. In all cases, it allowed correct identification of all human malaria parasite species.
Results were analysed by the signal to background ratio calculated by the array software analysis. The signal to background ratio for probes 'expected on' was always very significantly higher than for probes 'expected off', although the difference varied depending on the parasitaemia in the sample. To overcome this difficulty, ratios were expressed relative to the PCR control probe and analyses were performed using this methodology.
For drug resistance, the assay was able to detect the key mutation in Pfcrt (K76T) necessary for resistance to chloroquine, as well as the initial key mutation (PfhdhfrS108N) responsible for resistance to antifolate drugs such as pyrimethamine), the additional mutation conferring increased resistance (PfdhfrC59R) and the mutation conferring very high level resistance to these drugs (PfdhfrI164L). The assay gave variable results for the detection of a key mutation for resistance to proguanil (PfdhfrA16S/V) and for detection of mutations at another codon responsible for intermediate resistance to pyrimethamine (PfdhfrI51N). Finally the LoC assay was able to detect the wild type allele of Pfcytb268Y where mutations (Pfcytb268S/C) caused resistance to atovaquone.
This integrated PCR microarray LoC represented an innovation for its ease of use and cost effectiveness. The developed platform was the first semi-automated platform able to perform the diagnosis of the two major poverty related diseases and was easily adaptable for additional diagnostics purposes. The main features of platform made it suitable for the use in reference and regional laboratories also in low and middle income countries with high burden of tuberculosis and malaria.
The developed test could reliably identify, both from clinical samples and strains and on the same chip TB complex, main non TB species and the most frequent mutations leading to the MDR phenotype with high sensitivity and specificity. A chip for the XDR phenotype was also developed but not fully validated. Less frequent mutations (L533P, H526D, S315T variation two) were identified by the absence of hybridisation with the wild type probes.
The malaria assay allowed the specific identification of all human plasmodium parasite species. In addition the LoC could reliably detect drug resistant parasites carrying the acknowledged resistance mutations in Pfcrt, Pfdhfr and Pfcytb.
The proposed technology could be widely adopted, especially in TB and malaria endemic countries where it could have significant impact on morbidity and mortality of these diseases.
The scientific work conducted under this project led to several publications in international scientific journals, such as 'Antimicrobial agents and chemotherapy' and the 'Journal of clinical microbiology' by the American Society for Microbiology (ASM), the 'Journal of antimicrobial chemotherapy' by Oxford University Press, the 'BMC infectious diseases' and the 'International journal of antimicrobial agents'. During project implementation period, results of the scientific work were also presented during scientific congresses at national, European and international levels, in particular during national conferences and meetings and the annual congresses of the ASM and of the European Society of Mycobacteriology, that were held in several countries in Europe and in the United States of America. The project website was also used and constantly updated to disseminate main findings and project achievements.
A project website at 'http://www.tm-rest.org' was developed at the beginning of the project and was composed of a public section and a restricted area, accessible by project partners only for reporting relevant project information. The website was regularly maintained and updated with most relevant progress on research activities for dissemination to the public. A database of mutations conferring resistance to antimycobacterial first and second line drugs developed by P2-FZB with contribution from project partners was also posted in the website.