  patient 1 , the index case - patient , was a 66-year - old tunisian man with a 4-year history of untreated diabetes mellitus . during march 20april 27 , 2013
, he visited his daughter ( patient 2 ) in qatar for 5 weeks ( figure 1 ) , 1 week of which they spent on a muslim pilgrimage to mecca , kingdom of saudi arabia . on april 18 ,
results of a physical examination ( including chest radiograph ) for a visa extension in qatar were unremarkable . on the day of arrival back in tunisia ( april 28 ) , the patient experienced chills , followed by arthralgia , dry cough , and fever .
the daughter reported that her father had had no direct contact with camels during his stay in qatar or saudi arabia .
one of his children ( patient 3 , a nurse ) gave him acetaminophen and aspirin for 3 days and then intravenously administered dexamethasone ( 4 mg ) twice a day for 2 days . on may 6 ,
patient 1 experienced worsened dyspnea and he sought care at the centre hospitalier - universitaire fattouma bourguiba hospital ( monastir , tunisia ) emergency department , where he received a fifth injection of dexamethasone .
the patient was first admitted to the pulmonary ward , where he received amoxicillin - clavulanate ( 1 g ) 3 times daily ; however , on may 8 , respiratory failure and peripheral signs of shock necessitated admission to the intensive care unit ( icu ) , where he was positioned prone and given noradrenalin infusion and mechanical ventilation with additional nitric oxide .
clinical course of disease for patients with confirmed middle east respiratory syndrome coronavirus infection , tunisia , 2013 .
rh , regional hospital ; dh , district hospital ; rrt - pcr , real - time reverse transcription pcr .
mini ( < 10 ml fluid injected ) bronchoalveolar lavage recovered a liquid of low cellularity ; cultures for bacteria and fungi were negative .
the lavage fluid was then tested in the tunisia national reference laboratory ( tnrl ) for mers - cov by using real - time reverse transcription pcr ( rrt - pcr ) upe ( region upstream of the e gene ) , open reading frame ( orf ) 1a , and orf1b assays .
on may 10 , patient 1 died of multiple organ failure . because nasopharyngeal swab samples from his 2 adult children were positive for mers - cov , the case of patient 1
was reported to the world health organization as probable mers - cov infection ( 4 ) . on august 5 , 2013
, the centers for disease control and prevention ( cdc ) tested a serum sample collected from the index - patient on may 9 .
independent rrt - pcrs were positive for mers - cov ( 5 ) ; targets were upe ( cycle threshold [ ct ] 30.27 ) and nucleocapsid protein ( n)2 ( ct 30.46 ) .
sequences of the full n and spike ( s ) protein coding regions were submitted to genbank ( accession nos . kf811035 and kf811036 , respectively ) .
nucleotide / predicted amino acid sequence identities with published mers - cov sequences for the n and s gene coding regions ranged from 99.2%100% to 99.0%100% and from 99.4%99.9% to 99.4%99.8% , respectively .
phylogenetic relationships between this virus ( designated tunisia - qatar_2013 ) and other published mers - cov sequences showed clustering with geographically diverse sequences from saudi arabia and the united arab emirates ( figure 2 ) .  
midpoint - rooted phylogenetic trees of the full - length nucleocapsid ( n ) ( panel a ) and spike ( s ) ( panel b ) open - reading frames ( orfs ) of isolates obtained from index case - patient with middle east respiratory syndrome coronavirus ( mers - cov ) infection , tunisia , 2013 .
serum and available nucleotide sequences from genbank and public health england ( http://www.hpa.org.uk/webw/hpaweb&hpawebstandard/hpaweb_c/1317136246479 ) and the institut fr virologie ( http://www.virology-bonn.de/index.php?id  =  46 ) .
the estimated neighbor - joining trees were constructed from nucleotide alignments by using mega version 6.06 ( http://www.megasoftware.net ) .
numbers in parentheses denote the number of additional sequences from viruses isolated from humans that are identical to the listed sequence .
bootstrap support values ( 1,000 replicates ) 75% were plotted at the indicated internal branch nodes .
patient 2 was the 30-year - old daughter who had accompanied the index case - patient to mecca .
she remained in qatar until she attended her father s funeral in tunisia on may 11 , 2013 , when she reported sore throat , cough , and fever . on may 13 ,
a nasopharyngeal swab sample collected on may 16 was positive for mers - cov by rrt - pcr performed at the tnrl : upe ct 27.5 , orf1a ct 27.46 , and orf1b ct 37.55 .
testing at cdc detected a ct of 28.46 for upe and negative results for n2 and n3 ( 5 ) .
patient 3 was the 34-year - old son of the index case - patient , a nurse in the icu where his father had been admitted .
he had not traveled outside the country during the incubation period , and his first contact with the index case - patient was after his father s return to tunisia and illness onset .
he cared for his father at home during the initial phase of illness and thereafter in the pulmonology department and icu .
patient 3 reported a sore throat on the day after his father s funeral . a nasopharyngeal swab sample obtained on may 16 was positive for mers - cov by rrt - pcr performed at tnrl : upe ct 21.56 , orf1a ct 27.6 , and orf1b ct 31.39 .
at cdc , the nasopharyngeal swab sample was positive for mers - cov by 3 independent rrt - pcrs ( 5 ) : ct 21.67 for upe , 34.51 for n2 , and 32.32 for n3 .
nasopharyngeal and/or throat swab samples were collected a mean of 5 weeks after contact from the other 2 ( not ill ) children of patient 1 , his spouse , and the spouse of patient 3 .
health care workers who had been in contact with the index case - patient in the pulmonology ward ( n = 2 ) or icu ( n = 6 ) and who had reported sore throat , hyperthermia , and/or diarrhea ( 1 worker ) were also investigated . all respiratory samples from contacts were negative for mers - cov by rrt - pcr .
the fact that the diagnosis for the index case - patient was made by pcr of a serum sample collected 10 days after symptom onset and tested several weeks later highlights the value of testing serum samples for mers - cov rna .
this finding also provides valuable information about viremia in mers cov  infected patients , contributing to our understanding of the natural history of mers - cov infection and kinetics of virus shedding ( 7 ) .
given the incubation period of the disease ( up to 15 days ) , the father most likely acquired his infection in qatar ( 8,9 ) .
patient 3 , who had not traveled outside tunisia , could have been exposed during the 11 days he cared for his father at home and in the hospital .
the history of patient 2 is less clear ; she might have acquired the virus from the same source as her father in qatar , or she might have been secondarily infected by contact with him before he left qatar , given that her illness began almost 12 days after her father s .
patient 1 was severely ill at the time of icu admission ; in <3 days , his condition rapidly evolved to multiple organ system failure and death .
although we can not account for the diabetes or corticosteroid contributions to his disease severity , we can speculate that they might have worsened his outcome .
other mers cov patients who have died had concurrent conditions ( 2 ) , and corticosteroids are thought to worsen the outcomes for patients with influenza a(h1n1 ) virus infection ( 10 ) . 
 
the contact tracing results shed light on the potential for person - to - person transmissibility of mers - cov .
only 2 family members who had been in close and prolonged contact with the index case - patient became infected .
infection was not acquired by the case - patient s wife , his 2 children who did not live with him , or the icu workers who had short - term close contact with him .
however , these results should be interpreted cautiously because only nasopharyngeal swab samples obtained 5 weeks after contact with the index case - patient were tested .
in addition , serologic testing , which was not performed in the present investigation , could have shed more light on person - to - person mers - cov transmissibility .
chest radiograph of the middle east respiratory syndrome index case - patient , taken at the time of admission to intensive care unit ( may 8 , 2013 ) , tunisia .
 in 2013 in tunisia , 3 persons in 1 family were infected with middle east respiratory syndrome coronavirus ( mers - cov ) . 
 the index case - patient s respiratory tract samples were negative for mers - cov by reverse transcription pcr , but diagnosis was retrospectively confirmed by pcr of serum . 
 sequences clustered with those from saudi arabia and united arab emirates . 
The Cases
Conclusions
None