Interlaboratory evaluation of different extraction and real-time PCR methods for detection of Coxiella burnetii DNA in serum.

In the Netherlands, there is an ongoing and unparalleled outbreak of Q fever. Rapid and reliable methods to identify patients infected with Coxiella burnetii, the causative agent of Q fever, are urgently needed. We evaluated the performance of different DNA extraction methods and real-time PCR assays that are in use in seven diagnostic or reference laboratories in the Netherlands. A low degree of variation in the sensitivities of most of the developed real-time PCR assays was observed. However, PCR assays amplifying short DNA fragments yielded better results than those producing large DNA fragments. With regard to DNA extraction, the automated MagNA Pure Compact system and the manual QIAamp DNA mini kit consistently yielded better results than either the MagNA Pure LC system and NucliSens EasyMag (both automated) or the High Pure viral nucleic acid kit (manual). The present study shows that multiple combinations of DNA extraction kits and real-time PCR assays offer equivalent solutions to detect C. burnetii DNA in serum samples from patients suspected to have Q fever.

Clinical correlates of herpes simplex virus type 1 loads in the lower respiratory tract of critically ill patients.

BACKGROUND: The significance of isolation of herpes simplex virus (HSV) type 1 from the lower respiratory tract in critically ill patients on mechanical ventilation is still unclear. In the current study, we used polymerase chain reaction techniques to quantify HSV-1 to further evaluate its role. OBJECTIVES: The hypothesis was that high loads reflect invasive pulmonary disease related to prolonged mechanical ventilation and increased mortality, as opposed to shedding from the upper respiratory tract, which leads to lower viral loads. STUDY DESIGN: We prospectively studied 77 consecutive patients admitted to the intensive care unit and analyzed 136 tracheal aspirates or bronchoalveolar lavage fluids, taken when clinically indicated in the diagnostic workup of fever, radiologic pulmonary infiltrates, progressive respiratory insufficiency or combinations. Samples were cultured for bacteria and yeasts according to routine microbiological methods and HSV-1 loads were determined by real time quantitative PCR. Viral loads were expressed per number of cells recovered. RESULTS: HSV-1 load was directly related to the simplified acute physiology score II (rs=0.47, P=0.04) when the first specimen taken proved positive for HSV-1. HSV-1 positivity concurred with Candida spp. colonization. Patients with and without a HSV-1 load did not differ with respect to pulmonary and systemic courses and vital outcomes. CONCLUSIONS: The data suggest that HSV-1 in the lower respiratory tract originates from shedding in the upper respiratory tract in about 30% of critically ill patients, following immune suppression and reactivation, without invasively infecting the lung. No attributable mortality was observed.