16S rRNA Gene Sequence-Based Identification of Bacteria in Automatically Incubated Blood Culture Materials from Tropical Sub-Saharan Africa.
BACKGROUND
The quality of microbiological diagnostic procedures depends on pre-analytic conditions. We compared the results of 16S rRNA gene PCR and sequencing from automatically incubated blood culture materials from tropical Ghana with the results of cultural growth after automated incubation.


METHODS
Real-time 16S rRNA gene PCR and subsequent sequencing were applied to 1500 retained blood culture samples of Ghanaian patients admitted to a hospital with an unknown febrile illness after enrichment by automated culture.


RESULTS
Out of all 1500 samples, 191 were culture-positive and 98 isolates were considered etiologically relevant. Out of the 191 culture-positive samples, 16S rRNA gene PCR and sequencing led to concordant results in 65 cases at species level and an additional 62 cases at genus level. PCR was positive in further 360 out of 1309 culture-negative samples, sequencing results of which suggested etiologically relevant pathogen detections in 62 instances, detections of uncertain relevance in 50 instances, and DNA contamination due to sample preparation in 248 instances. In two instances, PCR failed to detect contaminants from the skin flora that were culturally detectable. Pre-analytical errors caused many Enterobacteriaceae to be missed by culture.


CONCLUSIONS
Potentially correctable pre-analytical conditions and not the fastidious nature of the bacteria caused most of the discrepancies. Although 16S rRNA gene PCR and sequencing in addition to culture led to an increase in detections of presumably etiologically relevant blood culture pathogens, the application of this procedure to samples from the tropics was hampered by a high contamination rate. Careful interpretation of diagnostic results is required.
Department of Tropical Medicine at the Bernhard Nocht Institute, German Armed Forces Hospital of Hamburg, Hamburg, Germany; Institute for Medical Microbiology, Virology and Hygiene, University Hospital Rostock, Rostock, Germany.
Hagen
H
Frickmann
Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany.
Denise
D
Dekker
Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany.
Norbert Georg
NG
Schwarz
Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany.
Andreas
A
Hahn
Kumasi Centre for Collaborative Research, Kumasi, Ghana.
Kennedy
K
Boahen
Kumasi Centre for Collaborative Research, Kumasi, Ghana.
Nimako
N
Sarpong
Kwame Nkrumah University of Science and Technology, Kumasi, Ghana.
Yaw
Y
Adu-Sarkodie
Laboratory Department 1, Central Institute of the German Armed Forces Medical Services Kiel, External site Berlin, Berlin, Germany.
Eva
E
Halbgewachs
International Vaccine Institute (IVI), Seoul, Korea.
Florian
F
Marks
International Vaccine Institute (IVI), Seoul, Korea.
Vera
V
von Kalckreuth
Department of Medical Microbiology at the University Medical Centre Gießen, Justus-Liebig University Gießen, Gießen, Germany.
Sven
S
Poppert
Department of Clinical Chemistry, University Medical Center Goettingen, Goettingen, Germany.
Ulrike
U
Loderstaedt
Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany.
Jürgen
J
May
Department of Tropical Medicine at the Bernhard Nocht Institute, German Armed Forces Hospital of Hamburg, Hamburg, Germany.
Ralf Matthias
RM
Hagen
D016470:Bacteremia / Q000175:diagnosis* / Q000382:microbiology; D001419:Bacteria / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D001431:Bacteriological Techniques / Q000379:methods / Q000592:standards; D004269:DNA, Bacterial / Q000235:genetics; D004275:DNA, Ribosomal / Q000235:genetics; D005869:Ghana; D006801:Humans; D058889:Molecular Typing / Q000379:methods; D012336:RNA, Ribosomal, 16S / Q000235:genetics*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000379:methods*
D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D012336:RNA, Ribosomal, 16S