Human Adenovirus Type 7 Outbreak in Police Training Center, Malaysia, 2011

In March 2011, an outbreak of acute respiratory disease was reported at the Kuala Lumpur (Malaysia) Police Training Centre. Approximately 100 trainees were hospitalized and 5 were admitted to the intensive care unit. Three of these 5 trainees died. Human adenovirus type 7 was identified as the etiologic agent.


The Study
In April 2011, the virology unit at the Institute for Medical Research, Kuala Lumpur, received respiratory samples from police trainees admitted to Kuala Lumpur Hospital with ARD and tissue samples from 2 of the 3 patients with fatal cases.The postmortem specimens consisted of cerebrospinal, pericardial effusion, and pleural effusion fl uids; lung, liver, spleen, and kidney tissues; skin; and bone marrow aspirate.
Viral nucleic acid was extracted from the clinical samples by using Roche High Pure Viral Nucleic Acid Kit (Roche Applied Science, Mannheim, Germany).Of the initial samples, 10 were screened for respiratory syncytial viruses, infl uenza viruses, parainfl uenza viruses, human metapneumoviruses, coxsackieviruses, echoviruses, rhinoviruses, coronaviruses, adenoviruses, and bocavirus by multiplex PCR.All samples were then subjected to adenovirus nucleic acid detection by PCR.
Partial HAdV hexon gene was amplifi ed by PCR (8).The SeqMan and Megalign software modules in the Lasergene suite of programs (DNASTAR, Madison, WI, USA) were used to format the nucleotide sequences.A phylogenetic tree was constructed by using the neighborjoining method from the MEGA4 software (www.megasoftware.net).
A total of 33 clinical specimens, including respiratory and fecal samples as well as postmortem samples, from 21 trainees admitted to Kuala Lumpur Hospital were collected.Of these, only 31 samples from 19 trainees were suffi cient for analysis.PCR or reverse transcription PCR was performed on 10 of the 31 samples by using primer sets specifi c for respiratory viruses with the ResPlex II Panel (QIAGEN, Valencia, CA, USA).The 10 samples contained HAdV-B species.
On the basis of this fi nding, partial hexon genes of adenovirus were amplifi ed from all 31 samples by using PCR with HAdV-specifi c primers (8).The results (Table ) indicated that 53% (10/19) of the samples tested were positive for adenovirus.An antemortem tracheal aspirate sample, received from the fi rst case-patient who died, was positive for adenovirus.Subsequently, postmortem lung and spleen tissue samples tested by PCR were also positive for adenovirus.In the second fatal case, adenovirus was detected from postmortem samples consisting of pleural effusion fl uid, pericardial effusion fl uid, lung tissue, and serum, but other tissues, such as heart, spleen, and liver, and cerebrospinal fl uid were negative (Figure 1).
All positive samples were sequenced, and BLAST sequencing analysis (http://blast.ncbi.nlm.nih.gov/Blast.cgi) showed that their sequences were similar to human adenovirus type 7 strain 0901H2/Shix/CHN/2009 isolated in People's Republic of China in 2009 (9).The phylogenetic tree, constructed on the basis of partial hexon gene (160) nucleotides (Figure 2), revealed that all positive samples by PCR belong to the species Human adenovirus B and are in the same cluster with adenovirus 7.

Conclusions
The outbreak of ARD, caused by HAdV-7, in the Kuala Lumpur Police Training Centre started in early March 2011.The police trainees had signs and symptoms of ARD such as fever, cough, and loss of appetite.The disease rapidly spread among the trainees, and communityacquired pneumonia was the initial diagnosis early in the outbreak.Other etiologic agents that recently caused ARD, including seasonal infl uenza virus and pandemic (H1N1) 2009 virus, were excluded because the reverse transcription PCR for infl uenza viruses was negative.
The source of the infection is not known.There is a strong possibility that one of the trainees was infected with HAdV-7 in the community outside the training center and then spread the infection to others once training resumed.Transmission occurs through respiratory droplets and close contact, which leads to rapid and widespread dissemination.Similar events have been seen in military camps (4), high schools, and day care centers (10).All affected trainees were 20-26 years old.Risk factors such as overcrowding, increased physical activities, and psychological stress   could possibly increase susceptibility to ARD.These risk factors associated with outbreaks of HAdV-caused ARD, which are prevalent in military and police training centers, have been reported in previous HAdV outbreaks in military recruits (11).In Malaysia, 2 researchers (12,13) described the role of HAdV in causing ARD (12).They analyzed 27 HAdV isolates from patients with ARD who sought consultation and treatment at University of Malaysia Medical Center during 1999-2005.Among the 27 isolates, the following species were represented: 19 (70%) belonged to HAdV-C, 6 (22%) belonged to HAdV-B, and 2 (7%) belonged to HAdV-F.Among the HAdV-B species isolates, 5 had the HAdV-3 serotype and only 1 had the HAdV-7 serotype.An earlier analysis of HAdV isolates in Malaysia ( 13) revealed that HAdV-21 was associated with acute fl accid paralysis during the outbreak of hand, foot, and mouth disease in Sarawak, but none of the isolates were HAdV-7.
Pneumonia caused by HAdV-7, commonly associated with lower respiratory tract infection, can lead to severe disease and death in some infants and immunocompromised persons (9,14).Outbreaks of pneumonia caused by HAdV-7 have been reported among hospitalized children in South Korea and in the United States (15).This outbreak demonstrated the potential of ARD caused by HAdV-7 to produce illness and death in police and army recruit camps and in institutional settings.HAdV-7 infection also is fatal in children.

Figure 2 .
Figure 2. Phylogenetic tree of partial hexon gene sequences (160 bp) of human adenovirus inferred by using the neighbor-joining method from the MEGA4 software (www.megasoftware.net).Study was in a police training center, Kuala Lumpur, Malaysia, 2011.The evolutionary distances were computed by using the maximum composite likelihood method.Species A-F are indicated by square brackets with duck adenovirus A as an outgroup.Thirteen human adenovirus isolates from the police training center outbreak are indicated in boldface.Representative strains of each species obtained from GenBank are labeled by using the adenovirus species and accession number.Bootstrap values (>75%) for 1,000 pseudoreplicate datasets are indicated at branch nodes.Scale bar indicates nucleotide substitutions per site.