Molecular epidemiology and significance of a cluster of cases of CMV infection occurring on a special care baby unit

Journal of Hospital Infection (1996) 34, 183-189

Molecular epidemiology and significance cluster of cases of CMV infection occurring special care baby unit C. Aitken,

J. Booth*, M. Booth*, Y. Boriskin*, S. Kempleyt and J. Breuer

of a on a

H. Testardj-,

Department of Virology and j-Department of Paediatrics, Royal Hospitals Trust, London El 1BB; *Department of Medical Microbiology, St George’s Hospital Medical School, London SW1 7 ORE, UK Received 10 January

1996; revised manuscript accepted 26 June 1996

Summary: Sequencing of the amino-terminal region of the glycoprotein H gene was used to investigate the possibility of a common source for cytomegalovirus (CMV) infection occurring in five premature babies. All but two of the strains were different, indicating that this cluster of infection occurring on the special care baby unit was unlikely to be epidemiologically linked.

Keywords: molecular

Cytomegalovirus epidemiology.

(CMV);

special

care

baby

unit

(SCBU);

Introduction In the United Kingdom, some 0*3-0*5% of babies are born congenitally infected with cytomegalovirus (CMV).‘s2 Most result from primary infection during pregnancy, the remainder from reinfection or reactivation of endogenous virus in the mother during gestation. Both peri- and post-natal CMV infection can also occur; the outcome is benign unless the child is premature or immunocompromised, in which case there is the possibility during infancy is common, with S-10% of respiratory disease.3s4 Infection of children under the age of five excreting CMV in their urine.’ Sources of infection include the mother’s genital tract, breast milk, saliva, and transfusions of CMV antibody-positive blood. Hospital-acquired infection has been demonstrated by restriction fragment length polymorphism.’ Cross-infection between children and nursing staff in child care facilities has been shown by similar methods, usually in those over two years old, raising the possibility of transmission to pregnant women.6 Correspondence London EClA 0195-6701/96/110183+07

to: Dr C. Aitken, 7BE. UK.

Virology

Clinical

Group,

St Bartholomew’s

Hospital,

West Smithfield,

0 1996 The Hospital

$12.00/O

183

Infection

Society

184

C. Aitken

et al.

The present investigation stemmed from an unusual cluster of symptomatic cases of CMV infection occurring in a special care baby unit (SCBU). To see if these arose from a contaminated common source, such as a blood donor, or by cross-infection, the viruses were typed by DNA sequencing and comparison of the predicted amino-acid sequence at the amino-terminal portion of the glycoprotein H protein. Chou7 demonstrated that this technique could be used to subdivide CMV isolates into two groups: group 1 resembled the prototype strain AD169 and group 2 resembled strain Towne. In our hands, the variability of this region was sufficient to demonstrate that the strains of CMV associated with the supposed outbreak were heterogeneous. Materials

and methods

CMV serology Serum samples from both mothers (antenatal booking sample) and babies were tested by commercial enzyme immunoassays for CMV total antibody (Centocor) and CMV IgM (Behring Diagnostics). All blood products were screened by the National Blood Transfusion Service Laboratories at Brentwood and retested as cases were identified.

CM V detection CMV was detected using two methods: by early antigen detection [detection of early antigen fluorescent foci (DEAFF)‘] using an FITC-conjugated monoclonal antibody to the 6 kDa immediate early antigen (TCC Ltd) and by routine culture.

Extraction

of DNA Specimens were mixed with an equal volume of extraction buffer (100 nM tris, pH 8,10 mM EDTA, 0.5% sodium dodecyl sulphate) followed by 10 PL proteinase K solution (20 mg/mL) per 125 PL of sample and incubation at 60°C for 30 min. After two extractions with 500 PL phenol:chloroform:isoamyl alcohol, the DNA was precipitated with two volumes of ethanol for 15 min at -7O”C, washed with 70% ethanol, dried under vacuum, then resuspended in 20 PL 10 mM tris, pH 7.8, 1 mM EDTA and stored at - 20°C until required. Fifty microlitres of polymerase chain reaction (PCR) buffer (50 mM KCl, 10 mM tris pH 9,0*1% Triton X-100), 25 mM MgC12, 250 PM of each dNTP triphosphate, O-5 units of Thermus aquaticus Taq polymerase and O-3 ltL of each 20 pM oligonucleotide primer were mixed with 1 PL of specimen DNA then subjected to 30 cycles of amplification. (94°C) for 30 s (for 5 min 30 s during the first cycle), 57°C for 30 s and 72°C for 90 s (for 16 min 30 s in the final cycle). The outside primers: (Jane-l)-S’CTCCTTCTCTCGGGTGTAAC-3’ and (JCB-2) S-GTATTCCATATGCCTCGATG-3’ amplified a 357 bp fragment; nested PCR of the

CMV on a SCBU

185

first-round product with the internal primers (Mil-1) Y-CCTGGATCACGCCGCT-3’ and (JCB-1) S-TAACAGCACGGTCGTCAG-3’ amplified a 257 bp DNA fragment. Amplification was in a Perkin Elmer Cetus programmable temperature block. The PCR amplification products were separated by electrophoresis in 6% polyacrylamide gel and the required band of DNA recovered. Sequencing was carried out using the Sequenase 2-O kit (Amersham) with the nucleotides being resolved on 6% sequagel (National Diagnostics).

Results

Clinical aspects The Royal London Hospital serves an ethnically mixed community of 240 000 about 20% of which is of Bangladeshi origin. Of the 3500 deliveries per year, 265 babies are admitted to the SCBU. From March to July 1993 five babies (A-E) developed signs consistent with CMV disease. Three (A-C) were premature (~30 weeks gestation), and two were delivered at or near term. Four babies (A, B, D, E) were delivered by emergency caesarian section, either because of maternal complications (A, B) or fetal distress (D, E). Baby C was one of twins born vaginally. The clinical presentations included thrombocytopaenia (A and C), pneumonitis (B) and intrauterine growth retardation (E). Baby D developed encephalitis when two-months old; and was treated empirically with ganciclovir because CMV was the only infectious agent identified. However, the baby failed to improve and metabolic investigations revealed an inherited disorder of copper metabolism (Menkes Kinky Hair Syndrome) which results in degeneration of the central nervous system. Other clinical features are summarized in Table I. Virological features Booking samples were available from four mothers (A, C, D, E); all were positive for CMV IgG. IgM was detected in the sample from baby C’s mother and from babies A and C. Three babies had received blood products (Table II), all of which had been screened and were later confirmed as CMV negative. CMV was detected by DEAFF and cultured from the urine of all the babies and a nasopharyngeal aspirate from baby B (Table II). Molecular epidemiological features DNA was extracted from the urine of four babies (A, B, C and D) and tested by nested PCR using primers designed to amplify the 5’ terminal region of the glycoprotein H gene of CMV. For three babies (A, B and C) duplicate specimens of cultured virus together with a specimen of urine,

186

C. Aitken Table

I. Clinical

features

et al.

of the babies identified

Gestation (weeks)

in the CMV

cluster

Clinical indications for admission to unit

Baby

Date of birth

Blood products

A

27.11.92

27

Premature delivery BW = 808 g; maternal hypertension

B

4.11.92

26

C

28.5.93

27

D

3.7.93

40

E

14.7.93

38

Emergency caesarian-maternal antepartum haemorrhage and spontaneous rupture of membrane BW = 912 g Premature delivery BW = 960 g. Hyaline membrane disease Emergency caesarian for fetal distress; recurrent seizures and failure to thrive BW = Emergency caesarian for fetal distress BW = 1.7 kg.

17 units (all CMV negative) Multiple packed cells (all CMV negative) 10 packed 1 platelet (? status) None None

SW, birthweight. Baby C was one of a twin pregnancy, the other baby did not develop any clinical or laboratory compatible with CMV disease. Baby D had Menkes disease.

Table

II.

Baby (date of birth)

Results of investigations

and possible sources of CMV in the CMV cluster

Date first positive DEAFF

CMV

A 25.11.92

March

1993

April fll.92 C 28.5.93 D 3.7.93 E 14.7.93

July

1993 1993

October

1993

July 1993 (six days after birth)

CMVG CMVM CMVG CMVM CMVG CMVM CMVG CMVM N/T

+ + + + + + -

N/T, not tested; PC, packed cells; PT, platelets; CMVG, IGM; +, antibody detected; -, antibody not detected.

features

amongst the babies identified Risk factors for CMV acquisition

serology Mother (booking serum)

Baby

cells

CMVG+ CMVM N/T

-

CMVG CMVM CMVG CMVM CMVG CMVM

+ + + + -

Blood

products

Breast

17 PC

Yes

PC (multiple)

Yes

1OPC 1PT None

Yes

None

Yes

CMV total antibody;

CMVM,

fed

Yes

CMV specific

for baby B, a single specimen of cultured virus was tested. The band of product of DNA was purified by polyacrylamide gel electrophoresis and sequenced. The predicted amino acid sequences for all the specimens are

CMV on a SCBU Virus

Predicted

strain AD169 Towne

patient

A

tc tc urine

patient

B

tc tc urine

patient

C

tc tc urine

patient

D

tc

187 amino

acid sequence

10 20 30 40 MRPGLPPYLTVFTVYLLSHLPSQRYGADAASEALDPHAFHLLL ...... S .. I.LA.C.....L.S....E.I..P..xK ...... ...... S .. I.LA.C.....L.S....E.V..P..xK ...... S..I.LA.C.....L.S....E.V..P..xK ...... ...... ...... S .. I.LA.C.....L.S....E.V..P..xK ...... ...... S..I.LA.C.....L.S....E.I..P..xK ...... ...... S .. I.LA.C.....L.S....E.I..P..xK ...... S..I.LA.C.....L.S....E.I..P..xK ...... ...... ...... S ..... A ......... ; ........................................ ...... S ..... A ......... ...... S ..... A ......... Q .................... S..I.LA.C.....L.S....E.V..P..xK ...... ......

Figure 1. Amino-terminal amino acid sequences for CMV glycoprotein H. x = codon deletion in the Towne sequence compared with the AD169 sequence; M =start codon for the glycoprotein sequence; tc=sequence obtained direct from virus isolated in tissue culture from the patient’s urine or nasopharyngeal aspirate; urine=sequence obtained direct from virus in the patient’s urine.

shown in Figure 1 together with the published sequences for the same regions of the prototype strains AD169 and Towne. In those babies from whom three specimens were examined (A, B and C), the same predicted amino acid sequence was recorded for all three specimens. The predicted amino acid sequences for babies A and D were identical but different from the sequences for babies B and C, which differed from each other. The sequences for babies A, B and D were recognizable as belonging to CMV group 2 (Towne) the sequence for baby C clearly belonged to CMV group 1 (like AD169, there being no deletion of a proline at position 36). These findings indicated that the cluster of cases of CMV infection in the SCBU were unlikely to have emanated from a common source. Discussion

Peri- and post-natally-acquired CMV infection in premature babies can result in severe, sometimes fatal infection. Well-documented routes of infection include the maternal genital tract at delivery, breast milk or transfusion,’ particularly of unscreened blood products.” The cases we report here were clustered in time and space, in such a way as to raise the suspicion of an epidemiological link. In view of the age of the babies at presentation (four presented at or above two months) and the difficulty in obtaining maternal specimens (four of the five mothers spoke no English) investigating the causes of the infections was difficult. We, therefore, decided to concentrate on the babies’ viral isolates to exclude an epidemiological link. Using the method described by Chou,’ we were able to confirm that the

188

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et al.

strains were different. This was enough to exclude a common source or nosocomial transmission. Two strains (babies A and D) were identical at both the amino acid and nucleotide level (Figure l), however, the babies were not in the ICU at the same time, thus making nosocomial transmission unlikely. Further genetic diversity could have been sought by examining the glycoprotein B gene.” The source of CMV in these babies was not immediately obvious. All had received screened products and four were delivered by caesarean section thus reducing the contact time with genital secretions. Congenital acquisition is likely in Baby C because there were detectable IgM antibodies in the maternal booking sample, however, it cannot be excluded in the others because CMV was first detected when they were one-month old. It is difficult to establish conclusively the role played by CMV in the babies’ symptoms. Three had symptoms compatible with moderate CMV disease-two had thrombocytopaenia and the other had pneumonitis. CMV is known to increase the risk of bacterial infections and this may have contributed to a staphylococcal bacteraemia which developed in baby C. Similarly, with baby E, intrauterine growth retardation was noted on a routine ultrasound scan at 23 weeks of gestation. Subsequently, the baby was shown to be excreting CMV at six days of age, confirming that intrauterine transmission had occurred. The neurological symptoms of baby D were initially attributed to CMV; however the diagnosis was later revised. Severe disease may occur in premature infants following post-natal acquisition of CMV because of the reduced levels of neutralizing antibody as a result of prematurity, and because there is continuing loss of antibody in neonates.“*‘2 This may explain the clinical features in babies A, B and C. The other possible source is breast milk, which all the babies received over long periods of time; however this remains conjecture as no milk samples were tested. In conclusion, despite limited sequence variability in CMV glycoprotein H, this study confirms the work of Chou7 that the amino-terminal region can be used to broadly differentiate clinical isolates. The method is, therefore, of value in solving problems of potential nosocomial infection, especially where obtaining additional information or samples is difficult.

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