Cytomegaloviruses in stored urine specimens

Volume 73 Number 4

Brie[ clinical and laboratory observalions

and two days of age (P < 0.001). T h e values for cord blood and maternal blood are not statistically significantly different. DISCUSSION As others have shown, we found that in the full-term infant the peak uric acid level occurs at one day of age and by two days of age the uric acid level is essentially at or slightly below the maternal level. H o w ever, in contrast to data in the literature, we find statistically valid differences between cord and maternal blood uric acid levels in both low-birth-weight and fullterm infants. T h e appearance of the peak value of serum uric acid in the low birth-weight infant is at a higher and more prolonged level than is the peak in the full-term infant. Since we do not know when the uric acid level in the full-term infant would level off, we cannot make a valid comparison between low birth-weight and fullterm infants at one and three weeks of age. SUMMARY 1. There is a small but statistically significant difference between maternal and cord

Cytomegaloviruses in stored urine specimens A quantitative study Roger A. Feldman, M.D. ATLANTA,

GA.

6 11

serum uric acid levels in both low-birthweight and full-term infants. 2. T h e r e is a significantly greater elevation of serum uric acid levels in the l o w birth-weight than in the full-term infant, and this elevation occurs for a more prolonged period of time. 3. D a t a on normal values of uric acid levels in infants will be of value in detecting patients with the Lesch-Nyhan syndrome. REFERENCES 1. Lesch, M., and Nyhan, W. L. : A famiIial disorder of uric acid metabolism and central nervous system function, Am. J. Med. 36: 561, 1964. 2. Marks, J. F., Baum, J., Keele, D. K., Kay, J. L., and MacFarlen, A.: Lesch-Nyhan syndrome treated from the early neonatal period, Pediatrics. In press. 3. Christiansson, G., and Josephson, B.: The uric acid concentration in serum from children, newborn infants and mothers after delivery, Acta paediat. 49: 633, 1960. 4. Kessel, I., and Paulitzer, W. M.: Blood uric acid levels in mothers and infants at birth, Arch. Dis. Child. 35: 310, 1960. 5. Leone, A., Chiappe, S., Chiappe, F., and Toeeo, A.: Sul ricambio del1'acido urico in soggetti prematuri e immaturi, Ann. Ital. Pediat. 19: 1, 1966. 6. Caraway, W. T.: Determination of uric acid in serum by a carbonate method, Am. Clin. Path. 25: 840, 1955.

terial may significantly reduce the probability of virus isolation. A note 4 records a half-life of 15 hours at 3 ~ C. and 1 hour at 37 ~ C. for a tissue culture replicated virus strain. T h e present report concerns cytomegalovirus infectivity titers and loss of infectivity in original urine specimens at various storage temperatures and suggests the clinical usefulness of studying refrigerated material. M A T E R I A L S AND M E T H O D S

REPORTS ~-8 suggest that, in the laboratory diagnosis of cytomegalovirus infection, storage of original specimen maSEVERAL

From the Virology Section, National Communicable Disease Center, Public Health Service, U. S. Department o[ Health, Education, and Wel[are.

Virus-positive urine specimens were obtained from 14 healthy children and from mothers of two children with clinical neonatal cytomegalovirus infection. Urine specimens were also studied from 13 children with clinical illness or history

6 12

Brie[ clinical and laboratory observations

suggestive of cytomegalovirus infection; five children with splenomegaly and petechiae in the first 24 hours of life~; two children with obstructive jaundice during the first six weeks of life; three children with infantile spasmsS; one child with microcephaly, intracranial calcifications, hepatosplenomegaly, and retardation; one child with a history, compatible with antenatal cytomegalovirus infection, first studied at two years of age; and one child observed in a prospective study of cytomegalovirus infectio.n during pregnancy. 7 Urine specimens were put on wet ice immediately after collection, and 0.2 ml. of an antibiotic mixture of penicillin, streptomycin, neomycin, and amphotericin B, in final concentrations of 100 units, 100 rag., 40 mg., and 1 /~g per milliliter, respectively, was added to 3.0 ml. of urine. Specimens were then stored in an electric refrigerator at 4 ~ C., except as noted. A large volume of one urine specimen which had been transported with wet ice over 800 miles by bus was received 36 hours after collection. One portion of this specimen was mixed with an equal volume of a 50 per cent suspension of sorbitol in tissue culture maintenance fluid (final concentration 25 per cent sorbitol and 1 per cent calf serum) and another portion mixed with an equal volume of tissue culture maintenance fluid (final concentration 1 per cent calf serum). Aliquots of these mixtures, stored at 4 ~ -20 ~ and - 6 0 ~ C., were titrated at intervals. Titrations of infectivity of virus in uncentrifuged urine were performed in human embryo lung fibroblast tissue culture with 0.1 ml. of inoculum in each of four tubes per tenfold dilution. The fibroblast tissue culture was maintained on a modified Eagle's medium with 2 per cent calf serum; fluids were changed at 3 to 6 day intervals. Infectivity and points were determined by the Reed-Muench method 32 to 36 days after inoculation. Cytomegalovirus strains were characterized by slowly developing focal cytopathogenic effects in tissue culture, and typical

The ]ournal o/ Pediatrics October 1968

Table I. Infectivity titers of cytomegalovirus in urine specimens Titer*

Asymptomatic children and adults

Symptomatic children

0-0.9 1-1.9 2-2.9 3-3.9 4-4.9

4 10 8 2 0

1 3 6 6 6

*Loglo TCIDso per 0.1 ml.

intranuclear and juxtanuclear cytoplasmic inclusions were seen in infected tissue culture stained with May-Gruenwald-Giemsa stain. TITRATIONS

Infectivity titers of virus in 24 specimens obtained from 14 healthy children and two mothers of clinical subjects are recorded in Table I. Titers ranged from 10 ~ to 10 a'5 TCIDs0 per 0.1 ml. of urine, with most between 101-~ and 102.7 TCIDs0 per 0.1 ml. of urine. Eight of the specimens were collected two to six months after a previous titration. The infectivity titers of virus in these specimens differed by less than a factor of four from previous titers. T h e titers of virus in specimens obtained from 13 children with illnesses or histories compatible with cytomegalovirus infection are also recorded in Table I. Titers ranged from 101"8 to greater than 105.5 TCIDs0 per 0.1 ml. of urine. The highest titers were found in specimens collected in the first weeks of life. Specimens with titers greater than 105.0 TCID~0 per 0.1 ml. produced a diffuse cytopathic effect within 18 to 48 hours after inoculation. Six urine specimens were studied during storage at 4 ~ C. fo.r periods of from 27 to 94 days (Table I I ) . The measured rates of decrease in infectivity of these six specimens varied, but the half-life averaged 5 to 10 days. Results of titrations of aliquots of a urine specimen diluted in tissue culture maintenance fluid with and without sorbitol, after storage at 4 ~ -20 ~ and -60 ~ C., are shown

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Brie[ clinical and laboratory observations

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Table II. Loss of virus infectivity in urine stored at 4 ~ C. Specimen 1

Specimen 2

Specimen 3

.

Specimen 4

Specimen 5

Specimen 6

Days Days Days I Days Days aJter l a]ter a/ter l after alter TCID~o/ eollec- TCID~o/ coIlec- TCID.,o/ eoIIec- TCID,o/ eollee- TCID~o/ collee- TCID~J 0.l mI. lion 0.1 rot. lion O.l ml. lion 0.1 ml, lion 0.l ml. t~on 0.l ml.

~>5.5 )5.5 ~5.5 4.5 2.0

2 11 18 43 87

)5.5 5.3 )5.5 4.8 2.8

10 16 23 48 94

4.5 4.0 3.5 (0

0 9 16

4.0

0

3.5

28

87

2.0

72

Table I I I . Infectivity titer of virus in urine after storage in maintenance fluid with 1 per cent calf serum with (+) and without (-) 25 per cent sorbitol Storage temperature 4 ~ C.

Days

+

6 13 23 44 87 129

5.5* 4.5 4.0 3.5 3.0 2.0

I

-

5.5 4.8 4.5 3.3 2.0

Storage temperature -20 ~ C.

+D2.5 2.3 1.0 (1.0

<0 d0 (0 (0

Storage temperature -60 ~ C.

+14.8 4.0 4.5 4.0

5.0 3.8 4.5 3.8

~Loglo TCID~o per 0.1 mL

in Table I I I . There was a steady decline in infectivity of materials stored at 4 ~ C., with some slight effect of sorbitol. Materials stored at - 6 0 ~ C. also slowly lost infectivity, with and without sorbitol. At - 2 0 ~ C., holyever, infectivity was lost more quickly than at 4 ~ or - 6 0 ~ C., and urine without sorbitol lost all measurable infectivity in two weeks of storage. DISCUSSION

Characterization of cytomegalovirus strains in this study was based on observation of cytopathic changes in fibroblast tissue culture and typical histologic changes in the infected cells. These cytopathic changes, described by Weller and Hanshaw, 1 R o w e and co-workers, 2 MacAllister and co-workers, s and others, served only to group the agents isolated, and no further subgrouping was attempted.

1.3 0

0

Days alter coIleclion

5.5

1

3.8

57

27

T h e amount o~ infectious cytomegalovirus found in urine specimens f r o m clinical cases and from asymptomatic children and adults is similar to and in some instances greater than those reported by 2Vledearis2 Highest titers were found in specimens collected in the first weeks of life from children with clinical illness. T h e fall in infectivity in six urine specimens which h a d been stored at 4 ~ C., and to which only an antibiotic suspension had been added, was significantly slower than that reported for a tissue culture replicated virus strain at 3 ~ C. 4 Considering the virus titers found in urine specimens taken from clinical cases, there is clearly sufficient time for virus isolation even after storage of a sterile specimen of urine on wet ice for severM days to perhaps weeks after collection. T h e results of study of a stored urine specimen with and without sorbitol show increases in stability with sorbitol at - 2 0 ~ C., and some effect at both 4 ~ C., and - 6 0 ~ C. SUMMARY

Titration of cytomegalovirus in original urine specimens from asymptomatie children and adults showed tilers of from 10 ~ to 10a'5 TCIDs0 per 0.1 ml., with most between 101-~ and 102.7 TCID~0 per 0.1 ml. I n specimens from children with a clinical illness or history suggestive of cytomegalic inclusion disease, titration results ranged from 101.5 to greater than 10 ~.5 TCID~0 per 0.1 ml., with the highest titers seen in specimens collected during the first weeks of life.

6 14

Brief clinical and laboratory observations

A slow loss of infectivity (5 to 10 day halflife) was observed at 4 ~ C. in six urine spedmens to which only an antibiotic suspension had been added. Sorbitol added to the urine had some effect in reducing the loss of infectivity, especially in specimens frozen at - 2 0 ~ C. Urine specimens stored for days to weeks at 4 ~ C. may be of significant value in the virologic study of children with cytomegalovirus infection. REFERENCES

1. Weller, T. H., and Hanshaw, J. B.: Virologic and clinical observations on cytomegalic inclusion disease, New England J. Med. 266: I233, 1962. 2. Rowe, W. P., Hartley, J. E., Cramblett, I.i. G., and Mastrota, F. M.: Detection of human salivary gland virus in the mouth and urine of children, Am. J. Hyg. 67: 57, 1958.

Dermal ridge, patterns." Technique/or their study in human fetuses James R. Miller, P h . D . * VANGOUVER~ BRITISlC[ COLUMBIA~ GANADA

The Journal o/ Pediatrics October 1968

3. Elek, S. D., and Stern, H.: Cytomegalic inclusion body disease (an example of a common infection causing a rare disease), Brit. J. Clin. Pract. 16: 627, 1962. 4. Krugman, R. D., and Goodheart, C. R.: Human cytomegalovirus; thermal inactivation, Virology 23: 290, 1964. 5. Feldman, R. A.: Generalized petechiae and splenomegaly in the newborn: Six cases associated with cytomegalovirus infection. In preparation. 6. Feldman, R. A., and Schwartz, J. F.: Possible association of cytomegalovirus infection with infantile spasms, Lancet 1: 180, 1968. 7. Feldman, R. A.: Cytomegalovirus infection during pregnancy: A prospective study and report of six cases. In preparation. 8. McAllister, R. M., Straw, R. M., Filbert, J. E., and Goodheart, C. R.: Human cytomegalovirus: Cytochemical observations of intracellular lesion development correlated with viral synthesis and release, Virology 19: 521, 1963. 9. Medearis, D. N., Jr.: Observations concerning human cytomegalovirus infection and disease, Bull. Johns Hopkins Hosp. 114: 181, 1964.

develop techniques to pemfit formulation in fetuses of those dermal patterns which are useful in clinical studies of malformed individuals postnatally. 3, 5 Such techniques would permit more detailed examination of stillborn and spontaneously aborted fetuses and might lead to the diagnosis of conditions that would be of significance in counseling parents regarding the outcome o~ subsequent pregnancies. MATERIAL

PaEvlous STUDIES on the embryology of dermatoglyphic patterns have been primarily histologic and concerned with the embryogenesis of the volar p a d s and the dermal ridges? T o my knowledge no previous attempts have been made to devise techniques for the study of pattern formulations. T h e purpose of the present study was to From the Department of Anatomy, Kyoto University, Kyoto, Japan, and Division of Medical Genetics, Department o[ Paediatrics, University of British Columbia, Vancouver, British Columbia, Canada. e'Requests for reprints should be sent to Division o[ Medical Genetics, Del)t. o~ Paediatrics University oJ British Columbia, Vancouver, B. C., -~anada.

AND METHODS

The material used in the study comprised 217 specimens obtained from the H u m a n Embryo Center, Department of Anatomy, University of Kyoto. An outline of the methods by which the younger specimens Were obtained has been given by Nishimura and colleagues4; the older specimens were obtained by induced delivery by using bougie or metreurynter and in a small number of cases by spontaneous abortion. Three groups of fetuses were examined: 23 specimens aged appro~:imately five months to term which had been fixed in 10 per cent formalin; 86 specimens of two to four months' gestation fixed in 10 per cent formalin or