Setting and population
La Réunion is a small tropical island (2,512 km2), located in the South Western Indian ocean, 700 km east of Madagascar. Landscapes are very contrasted with a mountainous centre separating a humid “windward” east coast from a dry “leeward” west coast. The domestic animal populations are comprised of roughly 40,000 cattle, 30,000 goats and 2,000 sheep, mainly based in the West and the South microregions . Coastal areas are the most densely populated and host approximately 80% of the 816,000 residents.
Between May 1 and October 31, 2013, all pregnant women presenting at the regional perinatal health care centre of Saint Pierre for an unexplained early (< 12 weeks) or late (12 to 21 weeks) miscarriage, stillbirth (intrauterine foetal death ≥22 weeks) preterm birth (PTB, <37 weeks) or small-for-gestational age child (SGA, birthweight <10th percentile), were proposed the addition of a Q fever workup in addition to the usual data collection of a birth registry [12,13]. Women were enrolled either prospectively, when the APO event was suspected (e.g., preterm labour, poor growth of uterine height), or retrospectively, when the APO event had occurred.
Sera were tested using an indirect fluorescent antibody (IFA) assay with commercially available antigens for C. burnetii (C. burnetti I+II IFA IgG/IgM/IgAt®, Vircell, Grenade, Spain).
Seropositivity was defined as a phase 2 or phase 1 IgG titre ≥1:64 with or without phase 2/1 IgM≥1:48. Acute Q fever was defined as a high phase 2 IgG titre ≥1:256 (compatible with a recent or an active infection ), or detection of C. burnetii genome on miscarriage products and placentas. These conservative thresholds were chosen to fulfil the National Reference Centre requirements and minimize the false positives . Persistent Q fever was defined as a phase 1 to phase 2 IgG ratio>1 in the absence of IgM antibodies . Women were proposed serology follow-up to check for seroconversion (4-fold increase in titres between 2 paired samples) as done in standard care.
Bacterial DNA within birth products was detected by real-time polymerase chain reaction (PCR) amplification of the IS1111 region of the C. burnetti genome. The DNA extraction, preparation of plasmid standards, PCR assays and probe analysis were performed using Klee’s protocol . Primers and probes were designed using the Primer Express software® (Applied Biosystems, Darmstadt, Germany) and purchased from TIB Molbiol (Berlin, Germany).
Biological plausibility was defined as a positive RT-PCR or the seroconversion of phase 2 IgG. The relationship between exposure to Q fever and APOs was deemed causal when temporality and biological plausibility criteria were met and TORCH (Toxoplasma gondii, other infections, rubella, cytomegalovirus, and herpes simplex virus [HSV]-2 or neonatal herpes simplex) pathogens ruled out.
Cumulative incidence rates of APOs were measured per 1,000 pregnant women within the participant sample and extrapolated to the total number of APOs in the population observed during the study period using resampling weights based on demographics to minimize selection and misclassification biases (Supplemental file).
Miscarriage, stillbirth, preterm birth, small-for-gestational age as well as a composite outcome of all these APOs were compared according to C. burnetii exposure using chi2 or Fisher exact tests. In addition, incidence rate ratios (IRR) of each Q fever related APO were estimated using Poisson regression models for dichotomous outcomes with the robust variance option adjusted for hypertensive disorders, diabetes (gestational or pre-gestational), and maternal addictions (smoking or alcohol). Attributable risk percent (i.e., etiologic fractions) among the exposed and population attributable fractions were generated to estimate the contribution of C. burnetii exposure to APOs.
All these analyses were performed using Stata 14.2® (StataCorp, College Station, TX, USA). For all estimations, a P value <0.05 was considered signiﬁcant.