@article{fdi:010094404,
  title = {{D}evelopment and evaluation of a triplex real-time {PCR} assay for enhanced plague diagnostics in {M}adagascar},
  author = {{R}amasindrazana, {B}. and {R}ahalison, {Z}. {A}. and {G}authier, {P}hilippe and {M}ikaty, {G}. and {B}odoarison, {Z}. {I}. and {M}aminirina, {L}. {A}. and {R}ahajandraibe, {S}. and {R}andriamanantsoa, {M}. {G}. and {K}ayoko, {G}. and {M}anuguerra, {J}. {C}. and {L}e {G}uern, {A}. {S}. and {R}asamindrakotroka, {A}. {J}. and {R}ajerison, {M}.},
  editor = {},
  language = {{ENG}},
  abstract = {{B}ackground {T}he plague, caused by {Y}ersinia pestis, remains a critical public health issue, particularly in endemic regions like {M}adagascar. {R}apid and accurate detection of this pathogen is essential for effective outbreak management and timely intervention. {F}ollowing the urban plague outbreak of 2017, a new molecular diagnostic algorithm was developed and introduced into routine use. {H}owever, certain cases required combining real-time and conventional polymerase chain reaction ({PCR}) methods. {W}hile effective, this approach often delayed obtaining conclusive results, an issue that can hinder swift outbreak responses. {T}he aim of this study is to design and optimize a three-target real-time {PCR} assay (q{PCR}) for the detection of {Y}. pestis in clinical samples. {M}ethods {T}he assay targeted three genes: caf1, pla, and yop{M}, located on the plasmids p{MT}1, p{PCP}1, and p{CD}1, respectively. {C}onducted at the {I}nstitut {P}asteur de {M}adagascar ({IPM}), the study evaluated the assay using both pure bacterial cultures and clinical samples, including 50 bubonic aspirates and 50 respiratory specimens. {R}esults {U}sing bacteriology technique as the reference standard, the triplex q{PCR} demonstrated a sensitivity of 100% (89-100%) and a specificity of 82%. {T}he positive predictive value ({PPV}) was 73% and the negative predictive value ({NPV}) was 100% (91-100%). {T}he coefficient of agreement kappa was 0.74, with a p-value of <0.0001. {N}otably, the new assay resolved 100% of previously inconclusive cases from the duplex q{PCR} test targeting only pla and caf1.{R}esults {U}sing bacteriology technique as the reference standard, the triplex q{PCR} demonstrated a sensitivity of 100% (89-100%) and a specificity of 82%. {T}he positive predictive value ({PPV}) was 73% and the negative predictive value ({NPV}) was 100% (91-100%). {T}he coefficient of agreement kappa was 0.74, with a p-value of <0.0001. {N}otably, the new assay resolved 100% of previously inconclusive cases from the duplex q{PCR} test targeting only pla and caf1. {D}iscussion {W}hile a new plague diagnostic algorithm has been set up after the outbreak in 2017, the present study suggests a real-time {PCR} assay based on three genes to improve the speed and accuracy of plague diagnostic. {F}urthermore, this new technique is a valuable tool for managing plague outbreaks and supporting field diagnostics not only in {M}adagascar but also in countries with plague. {C}onclusions {T}he developed triplex assay to molecularly diagnose {Y}. pestis in human samples improves the standard already in place and allows to resolve ambiguities previously associated with inconclusive results from duplex q{PCR} tests, thereby reinforcing the reliability and accuracy of this new technique. {I}mplementing this new method into routine will enable a faster, more effective response to plague outbreaks by reducing the time needed to confirm plague cases and limiting the spread of the diseases. {T}his new technique is also flexible and can be undertaken close to human cases with adequate biosecurity and biosafety measures.},
  keywords = {{MADAGASCAR}},
  booktitle = {},
  journal = {{PL}o{S} {N}eglected {T}ropical {D}iseases},
  volume = {19},
  numero = {7},
  pages = {e0013278 [14 ]},
  ISSN = {1935-2735},
  year = {2025},
  DOI = {10.1371/journal.pntd.0013278},
  URL = {https://www.documentation.ird.fr/hor/fdi:010094404},
}