Journal of Infection (2009) 59, 366e370

www.elsevierhealth.com/journals/jinf

CASE REPORT

Fatal co-infection with swine origin influenzavirus A/H1N1 and community-acquiredmethicillin-resistant Staphylococcus aureus

Vincent C.C. Cheng a, Yuk-Kong Lau b, Kwok-Lun Lee b, Kwan-Ho Yiu c,Kwok-Hung Chan a, Pak-Leung Ho a, Kwok-Yung Yuen a,*

a Department of Microbiology, Queen Mary Hospital, The University of Hong Kong, Hong Kong, Chinab Department of Medicine, Ruttonjee & Tang Shiu Kin Hospitals, Hong Kong, Chinac Department of Pathology, Pamela Youde Nethersole Eastern Hospital, Hong Kong, China

Accepted 12 August 2009Available online 9 September 2009

KEYWORDSSwine origin influenzavirus A/H1N1;CA-MRSA;Pneumonia;Multiplex PCR

* Correspondence to: Carol Yu CentrInfectious Diseases, Department of MiHong Kong, Queen Mary Hospital, PokKong, China. Tel.: þ852 28553206; fa

E-mail address: kyyuen@hkucc.hku

0163-4453/$36 ª 2009 The British Infdoi:10.1016/j.jinf.2009.08.021

Summary Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA)co-infection has been described previously in association with respiratory tract infectioncaused by seasonal influenza A viruses, but not with swine origin influenza A (H1N1) virus(S-OIV). We report the clinical and pathological findings of the first death with fulminant co-infection by CA-MRSA. Since early empirical treatment with beta-lactam plus fluoroquinoloneor macrolides is often initiated before specimen collections, bacterial co-infection in S-OIVmay have been under-reported.ª 2009 The British Infection Society. Published by Elsevier Ltd. All rights reserved.

Introduction

Since the outbreak of the novel swine origin influenza A(H1N1) virus (S-OIV) in Mexico in February 2009, there havebeen more than 100,000 laboratory confirmed human casesreported from 124 countries to World Health Organizationas of 15 July 2009.1 The initial clinical manifestations of

e for Infection and Division ofcrobiology, The University offulam Road, Pokfulam, Hongx: þ852 28724555..hk (K.-Y. Yuen).

ection Society. Published by Elsev

S-OIV included asymptomatic infection, mild upper respira-tory tract infection with or without fever, and occasionalgastrointestinal symptoms of vomiting or diarrhea.2 Only2e6% of confirmed cases required hospitalization for lowerrespiratory symptoms due to viral pneumonia. The medianage of hospitalized patients was ranging from 15 to 42years.1 The reported deaths have occurred mostly inpatients aged between 20 and 49 years,3 which is lowerthan that of seasonal influenza.4 The median time fromsymptom onset to hospitalization was 6 days (range: 1e20days), whereas and the median time from symptom onsetto death was 10 days (range: 2e33 days).3 Preliminary his-topathological findings revealed diffuse alveolar and inter-stitial damage with lymphocytic infiltrates suggestive of

ier Ltd. All rights reserved.

Fatal co-infection with swine origin influenza virus A/H1N1 367

viral pneumonitis. Secondary bacterial pneumonia hasseldom been reported in autopsied cases.1 Here wedescribe the first case of death related to S-OIV infectioncomplicated by bacteremic pneumonia due to community-acquired methicillin-resistant Staphylococcus aureus(CA-MRSA) in an immunocompetent adult.

In Hong Kong, reporting of both S-OIV and CA-MRSAinfection is mandatory. Since the first case of S-OIV wasdocumented on 1 May 2009, there have been 2702 con-firmed case of S-OIV as of 26 July 2009. As of 31 May 2009,there are 586 cases of CA-MRSA infection reported to theCentre for Health Protection of the Department of Healthsince it became a notifiable disease in January 2007(http://www.chp.gov.hk/).

Case report

A 42-year-old previously healthy sailor attended thehospital emergency room on 8 July 2009 because of chestpain, cough for 5 days and dyspnea for 2 days. Histemperature was 37 �C, respiratory rate was 38/min, andoxygen saturation was 78% on room air. A chest X-rayshowed extensive bilateral consolidation and right-sidedpleural effusion. His total white cell and neutrophilcounts were 19.1� 109/l (normal range 3.8e10.0� 109/l)and 15.6� 109/l (normal range 1.8e6.7� 109/l) respec-tively. Toxic granules of neutrophils and atypical lympho-cytes were present in the peripheral smear. Thehemoglobin and platelet counts were 13.9 g/dL (normalrange 12.5e17.5 g/dL) and 252� 109/l (normal range150e400� 109/l) respectively. His serum creatinine,alanine transaminase, spot glucose levels, and creatininekinase were elevated to 264 mmol/l (normal range62e115 mmol/l), 122 IU/l (normal range< 41 IU/l),44 mmol/l (normal range of fasting glucose 3e6 mmol/l),and 1769 IU/l (normal range 24e180 IU/l), respectively. Di-agnostic pleurocentesis on the day of admission showed nu-merous white cells and Gram-positive cocci in clusters onGram stained smear. He was given vancomycin and clin-damycin in addition to the empirical therapy of piperacil-linetazobactam, and clarithromycin for severecommunity-acquired pneumonia (CAP). His condition rap-idly deteriorated with respiratory, circulatory and acuterenal failures, and required mechanical ventilation,inotropic support and hemodialysis. He succumbed 48 hafter admission despite maximum supportive treatment.Postmortem examination revealed evidence of mixed bac-terial and viral pneumonia (Fig. 1aed), and the diaphrag-matic skeletal muscle showing lots of polymorphsbetween muscle fibres. Both blood and pleural fluid cul-tures grew a MRSA which was sensitive to vancomycin,erythromycin, clindamycin, fusidic acid, gentamicin andcotrimoxazole. The MRSA isolates were analyzed furtherby PCR and sequencing as previously described.5,6 Theresults showed presence of a S. aureus-specific target,sau (108 bp), the PantoneValentine Leukocidin (PVL)genes, lukS-lukF (114 bp) and the staphylococcal cassettechromosome (SCC) mec type IV. Multilocus and spa se-quencing showed that the isolates exhibited the ST30 al-lelic profile and t019 type, respectively. These findingswere characteristic of the widespread CA-MRSA clone,

designated as HKU100 in our locality.5 Influenza A viralantigen detection on the nasopharyngeal aspirate (NPA)collected one day before the patient’s death was nega-tive. However, the same NPA sample (3.06� 107 copies/ml), the paramortem nasopharyngeal swab (2.24� 105

copies/ml) and the lung tissue (50 copies/ml) were allpositive for S-OIV by LightCycler and ultrarapid RT-PCRperformed as previously described.7 As the bacterial cul-tures of blood and pleural fluid were positive for CA-MRSAwhile the rapid antigen test, Directigen EZ Flu Aþ B (Bec-ton Dickinson) for S-OIV on NPA was negative initially, thediagnosis of S-OIV was not considered and oseltamivir wasnot given.

Method

A multiplex PCR specific for S-OIV H1, sau, and mecA was per-formed asmodified fromourpreviousprotocol.5,6 Briefly, totalnucleicacid from 250 ml ofNPA of this patient wasextractedbyusing the NucliSEn easyMAG (bioMerieux) and 12 ml of the ex-tracted total nucleic acid was used to prepare cDNA by Invitro-gen Superscript III kit with random primer as previouslydescribed.8 2 ml of cDNA was used for multiplex PCR amplifica-tion reaction with QIAGEN multiplex PCR master mix (QIAGENGmbH, Hilden, Germany) and 0.5 mM of each primer (mecAprimers: sense 50-TCCAGATTACAACTTCACCAGG-30 and anti-sense 50-CCACTTCATATCTTGTAA CG-30; Sau primers: sense50-AATCTTTGTCGGTACACGATATTCTTCACG-30 and anti-sense50-CGTAATGAGATTTCAGTAGATAATACAACA-30; S-OIV H1primers: sense 50-GGTAAATGTAACATTGCT-30 and anti-sense50-ACAATGTAGGACCATGAG CTT-30). Samples were amplifiedby heating at 95 �C for 15 min, 40 cycles of 94 �C for 30 s,55 �C for 30 s, 72 �C for 30 s, and a final of extension periodof 10 min at 72 �C. PCR products were analyzed by electro-phoresis in a 2% (w/v) agarose gel and stained with 0.5 mg/ml of ethidium bromide (Fig. 2).

Discussion

CA-MRSA co-infection has been described previously inassociation with respiratory tract infection with seasonalinfluenza A viruses,9 but not with S-OIV. We report the clin-ical and pathological findings of the first death withfulminant co-infection by a PVL-positive CA-MRSA in animmunocompetent adult, who succumbed 7 days after on-set of symptoms. PVL-positive S. aureus CAP is often char-acterized by extensive necrosis of lung tissue with a highcase-fatality. In a case series, six out of eight patientswith severe CAP caused by S. aureus carrying PVL gene suc-cumbed to the infection.10 Subsequently, the same group ofinvestigator comparing eight patients of CAP caused byPVL-positive S. aureus and another 36 cases caused byPVL-negative S. aureus, the former was found to occurmore frequently in immunocompetent children and youngadult and the survival rate 48 h after admission was signif-icantly lower for PVL-positive cases.11

Post-influenza staphylococcal pneumonia has been re-ported in healthy adult during the past influenza pan-demic,12 as influenza virus infection increases susceptibilityto bacterial respiratory pathogens in both in-vitro and

Figure 1 Histological examination of the lung tissue. (1a) Magnification (100�) showing diffuse alveolar damage & exudation.H&E stain: lung parenchyma showing varying degree of diffuse alveolar damage with hyaline membrane and some alveoli pluggedwith fibrin and fluid. (1b) Magnification (200�) showing changes typical of bacterial pneumonia. H&E stain: lung parenchymashowing patchy consolidation with polymorphs inside the alveolar spaces. Clusters of Gram-positive cocci are identified in thesubsequent Gram stain (not shown). (1c) Magnification (200�) showing changes suggestive of viral pneumonitis. H&E stain: alve-olar spaces with necrotic debris and detached pneumocytes. One of them is binucleated (arrow). (1d) Magnification (400�)showing changes suggestive of viral pneumonitis. H&E stain: atypical pneumocytes with mildly enlarged nuclei, granular to vac-uolated cytoplasm (arrow), compatible with viral pneumonitis.

368 V.C.C. Cheng et al.

in-vivo models.13,14 With the emergence of CA-MRSA in theUnited States where the national prevalence of nasal MRSAcolonization was 1.5% in year 2004,15 CA-MRSA pneumoniahas been reported in 15 (88%) of 17 immunocompetentcases of S. aureus CAP during a recent seasonal influenza(2003e2004) in which 5 (29%) out of 17 patients died ina median of 7 days after symptom onset.9

However, S-OIV and CA-MRSA co-infection has not beenreported in the literature since the outbreak of this novelinfluenza A virus in 2009. In the early report from Mexico,there were only 2 instances of co-infections (1 Streptococ-cus pneumoniae, 1 adenovirus) by molecular detectionamong 21 severe or fatal cases.3 Further investigation of18 confirmed S-OIV cases with pneumonia requiring hospi-talization at the National Institute of Respiratory Diseasesin Mexico City, none of them had co-infection with otherrespiratory viruses or atypical bacteria (Legionella pneu-mophila, Chlamydophila pneumoniae, and Mycoplasmapneumoniae) as detected by multiplex PCR.16 In theintensive-care patients with severe S-OIV in Michigan ofthe United States, none of the 10 patients had evidenceof bacterial co-infection in the lungs by culture of bron-choalveolar lavage.17 The lack of bacterial co-infection inpatients with S-OIV could be related to the empirical anti-biotic therapy including a combination of beta-lactam plusfluoroquinolones or macrolides for CAP before the

collection of clinical specimens. In the above mentionedcohort of 18 confirmed S-OIV cases with pneumonia, 12 pa-tients attended outpatient clinics before hospitalizationand were treated with one or more antibiotics, includingceftriaxone in five patients, amikacin in three, amoxicil-lineclavulanate in two, azithromycin in one, or other mac-rolides in three, or another antibiotics in two.16 Among 10patients in the intensive care unit (ICU) in Michigan, all re-ceived antibiotic therapy upon admission to the initial hos-pitals, and broad-spectrum antibiotics were continuedupon transfer to the ICU.17 Therefore, the incidence ofbacterial co-infection in S-OIV might be markedly under-reported at the present moment. However, with increasingnumber of S-OIV, co-infections due to S-OIV and CA-MRSAmight be observed in populations with high prevalence ofCA-MRSA colonization. In particular, the empirical antibi-otic treatment of CAP in immunocompetent adults withb-lactam antibiotics or respiratory fluoroquinolones maynot adequately cover CA-MRSA. In the cases of CA-MRSApneumonia reported during 2003e2004 seasonal influenza,all of the CA-MRSA isolates were uniformly resistant tomacrolides and half of them were nonsusceptible to fluoro-quinolones,9 whereas ciprofloxacin resistant PVL-positiveCA-MRSA has been sporadically reported in UK.18,19

In summary, a high level of clinical vigilance is requiredto manage patients with severe CAP in the era of pandemic

Figure 2 Multiplex PCR assay for detection of S-OIV H1, sau,and mecA. Patient and control samples were amplified by mul-tiplex PCR. Gel electrophoresis of these reactions is shown.Lanes 1 and 9: 1-kb DNA ladder markers, lane 2: patient nasalswab (postmortem), lane 3: patient’s initial nasopharyngeal as-pirate, lane 4: multiplex positive (S-OIV H1, sau, and mecA)control, lane 5: mecA positive control, lane 6: sau positive con-trol, lane 7: S-OIV H1 positive control, lane 8: water as nega-tive control. The sizes of S-OIV H1 gene, sau gene, and mecAgene PCR products were 83 bp, 108 bp, and 162 bp respec-tively. The turn-around time of the test is 6 hours.

Fatal co-infection with swine origin influenza virus A/H1N1 369

influenza. When necrotizing pneumonia caused byPVL-positive CA-MRSA is strongly suspected, especially inpatients with haemoptysis, radiographic appearance ofcavitatory lesions and pneumatoceles, or pleural fluid orbronchial aspirate lavage showing clusters of Gram-positivecocci, known exposure to or colonization of CA-MRSA, orconcurrent skin infection, specific antimicrobial againstCA-MRSA should be initiated. Although the 2007 IDSA/ATSguidelines recommend the addition of vancomycin or line-zolid for empirical treatment of CAP if CA-MRSA is a consid-eration,20 linezolid may be a better choice due to its goodpharmacokinetic profile in lung.21 As false negative resultsof the influenza A virus antigen test might occur due tothe low sensitivity (51%) of influenza nucleoprotein antigendetection,22 empirical antiviral agents such as neuramini-dase inhibitors should be considered. Alternatively, a multi-plex PCR (S-OIV H1, sau, and mecA) could be performed onthe respiratory specimens in patients with severe CAP todetect this highly fatal co-infection so that appropriate an-timicrobial regimen could be given to improve theoutcome.

Acknowledgement

The authors would like to acknowledge Dr Rodney Li andDr Alan Wu of the Department of Pathology, PamelaYoude Nethersole Eastern Hospital for their kind facilita-tion of specimen collection. The study is partly funded bythe Providence Foundation Limited in memory of the lateDr. Lui Hac Minh and the Research Fund for the Controlof Infectious Diseases (RFCID) of the Food and HealthBureau of the Hong Kong SAR Government.

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