The atovaquone resistance of malaria parasites correlates with mutations in the cytochrome b gene. We sequenced the Plasmodium falciparum cytochrome b gene of 135 African isolates. Our data showed a high mutation rate (8.9%); however, the risk of emergence spreading of atovaquone-resistant P. falciparum strains could be limited.

Clinical microbiology is experiencing the emergence of the syndromic approach of diagnosis. This paradigm shift will require innovative technologies to detect rapidly, and in a single sample, multiple pathogens associated with an infectious disease. Here, we report on a multiplex technology based on DNA-microarray that allows detecting and discriminating 11 bacteria implicated in respiratory tract infection. The process requires a PCR amplification of bacterial 16S rDNA, a 30 min hybridization step on species-specific oligoprobes covalently linked on dendrimers coated glass slides (DendriChips®) and a reading of the slides by a dedicated laser scanner. A diagnostic result is delivered in about 4 h as a predictive value of presence/absence of pathogens using a decision algorithm based on machine-learning method, which was constructed from hybridization profiles of known bacterial and clinical isolated samples and which can be regularly enriched with hybridization profiles from clinical samples. We demonstrated that our technology converged in more than 95% of cases with the microbiological culture for bacteria detection and identification.

Given the emergence of drug resistance and the high rate of polyclonal microorganism infections, the availability of a fast and sensitive test to detect minority mutant populations would be an improvement in the diagnosis of infectious diseases. A clamped-probe real-time PCR assay to diagnose the Plasmodium falciparum K76T mutation in clone populations was developed, using a wild-type-specific locked-nucleic-acid-containing oligomer to suppress wild-type PCR amplification and to enhance melting analysis with a mutation-specific detection probe.In the last few decades, the emergence of drug resistance has become a major problem for the treatment of various infectious diseases. Bacterial, viral, or parasitic infections are often polyclonal (4,10,12,25,27). Drug-resistant individuals may hide among a sensitive population and the identification of a resistant clone may have implications for epidemiology and treatment. Molecular diagnosis of resistant microorganisms by mutation detection is a routine examination in virology (17,20) and has started to be used in bacteriology (5,19,23) and parasitology (24). Recent virology studies have indicated that the presence of minority drug-resistant variants may be an independent predictor of treatment failure (8, 9). The emergence of nosocomial infections caused by drug-resistant bacteria also represents a significant threat for debilitated patients (22). As a consequence, a need exists to have a fast and sensitive diagnostic test to identify minority populations of resistant organisms. In order to improve the efficacy of such a genotype-assisted diagnosis, methods have been developed to lower the detection threshold of minority populations among a wild-type one. This trend was demonstrated by two recent studies. First, a rapid and accurate method of duplex real-time PCR used to detect ampicillin-resistant Enterococcus faecium, a major cause of nosocomial infections, was recently published (19). Second, an RNA heteroduplex generator tracking assay elicited a decrease in the detection threshold of type 1 human immunodeficiency virus mutant populations from 20 to 0.5% (12). Concerning the diagnosis of Plasmodium falciparum malaria, the detection of a minority population has become important with the discovery of Pfcrt K76T mutation, which has been correlated with in vitro chloroquine resistance (1,7,26).Chloroquine has proved to be one of the most successful drugs ever deployed against P. falciparum malaria, mainly because it is well tolerated and available at a very low cost. Over the last three decades, P. falciparum chloroquine resistance has significantly increased, and many countries have discarded this drug from the antimalarial weaponry. In these areas, it has recently been observed that the reappearance of chloroquinesensitive P. falciparum strains has followed chloroquine withdrawal (15, 21). This phenomenon, if confirmed in the future, could make reusing chloroquine as the first line treatment of malaria possible (18, 21), e.g., in combination with artemisinin derivati...

Research of ancient pathogens in ancient human skeletons has been mainly carried out on the basis of one essential historical or archaeological observation, permitting specific pathogens to be targeted. Detection of ancient human pathogens without such evidence is more difficult, since the quantity and quality of ancient DNA, as well as the environmental bacteria potentially present in the sample, limit the analyses possible. Using human lung tissue and/or teeth samples from burials in eastern Siberia, dating from the end of 17th to the 19th century, we propose a methodology that includes the: 1) amplification of all 16S rDNA gene sequences present in each sample; 2) identification of all bacterial DNA sequences with a degree of identity ≥95%, according to quality criteria; 3) identification and confirmation of bacterial pathogens by the amplification of the rpoB gene; and 4) establishment of authenticity criteria for ancient DNA. This study demonstrates that from teeth samples originating from ancient human subjects, we can realise: 1) the correct identification of bacterial molecular sequence signatures by quality criteria; 2) the separation of environmental and pathogenic bacterial 16S rDNA sequences; 3) the distribution of bacterial species for each subject and for each burial; and 4) the characterisation of bacteria specific to the permafrost. Moreover, we identified three pathogens in different teeth samples by 16S rDNA sequence amplification: Bordetella sp., Streptococcus pneumoniae and Shigella dysenteriae. We tested for the presence of these pathogens by amplifying the rpoB gene. For the first time, we confirmed sequences from Bordetella pertussis in the lungs of an ancient male Siberian subject, whose grave dated from the end of the 17th century to the early 18th century.

After the microbiology based on Pasteur's method and polymerase chain reaction (PCR), the diagnosis company named Dendris has proposed a third-generation of diagnosis enabling the search of a broad range of pathogens with strong sensitivity and specificity. This extraordinary profile was possible thanks to the use of phosphorus dendrimers for which various techniques of deposition on a given support were investigated and described and analyzed in this report.