Rapid detection and quantification of cell free cytomegalovirus by a high-speed centrifugation-based microculture assay: comparison to longitudinally analyzed viral DNA load and pp67 late transcript during lactation.
BACKGROUND
Human cytomegalovirus (HCMV) is reactivated in nearly every seropositive breastfeeding mother during lactation [Lancet 357 (2001) 513]. Conventional tissue culture (TC) and low-speed centrifugation-enhanced microtiter culture methods are not able to detect HCMV from milk during all stages of lactation.


OBJECTIVE
Development of a sensitive and quantitative microculture technique to describe the dynamics of HCMV reactivation in different milk compartments during lactation.


METHODS
Milk samples were collected longitudinally from seropositive breastfeeding mothers of preterm infants. Native milk samples were separated into fraction 1 (aqueous extract of milk fat), fraction 2 (cell and fat free milk whey) and fraction 3 (milk cells). Each of these fractions was screened qualitatively (TC, nPCR, pp67 late mRNA) and quantitatively (high-speed centrifugation-based microculture, quantitative PCR).


RESULTS
Prior to low-speed centrifugation-enhanced inoculation, virus particles were concentrated by high-speed centrifugation (60 min at 50,000 x g, 4 degrees C). Using fraction 2 we were able to describe the dynamics of viral reactivation during lactation. We present the course of the quantitative virolactia and DNAlactia and qualitative detection of HCMV pp67 late mRNA in milk whey of four mothers (three transmitters and one non-transmitter). In all these cases virolactia described an unimodal and self limited course. Peak levels of virolactia for transmitters (T1: day 44; T2: day 43; T3: day 50) were closely related the onset of viruria of the corresponding preterm infants (U1: day 39; U2a/U2b: day 44/57; U3: day 60). The courses of viral load coincidence with the courses of DNA load.


CONCLUSIONS
We present a rapid and highly sensitive microculture method for the quantification of cell free HCMV from milk whey and aqueous extracts from milk fat. Viral reactivation during lactation describes an unimodal course. Our findings have strong implications for quality control of any virus inactivation procedure.
Hamprecht
Klaus
K
Institute of Medical Virology and Epidemiology of Viral Diseases, University Hospital of Tübingen, Elfriede-Aulhorn-Str 6, 72076 Tübingen, Germany. kshampre@med.uni-tuebingen.de
Witzel
Simone
S
Maschmann
Jens
J
Dietz
Klaus
K
Baumeister
Andrea
A
Mikeler
Elfriede
E
Goelz
Rangmar
R
Speer
Christian P
CP
Jahn
Gerhard
G
D001942:Breast Feeding; D002498:Centrifugation; D003587:Cytomegalovirus / Q000235:genetics / Q000302:isolation & purification* / Q000502:physiology; D003586:Cytomegalovirus Infections / Q000635:transmission / Q000821:virology; D004279:DNA, Viral / Q000032:analysis; D005260:Female; D006801:Humans; D007231:Infant, Newborn; D007234:Infant, Premature; D007235:Infant, Premature, Diseases / Q000821:virology*; D018445:Infectious Disease Transmission, Vertical; D007774:Lactation / Q000378:metabolism; D008895:Milk, Human / Q000821:virology*; D010750:Phosphoproteins / Q000378:metabolism; D016133:Polymerase Chain Reaction; D015203:Reproducibility of Results; D013997:Time Factors; D014158:Transcription, Genetic; D019562:Viral Load; D014763:Viral Matrix Proteins / Q000378:metabolism; D014776:Virus Cultivation
D004279:DNA, Viral; D010750:Phosphoproteins; D014763:Viral Matrix Proteins; C464963:pp67 protein, human cytomegalovirus