Transactions of the Royal Society of Tropical Medicine and Hygiene (2005) 99, 577—584

Crimean-Congo hemorrhagic fever: experience ata tertiary care hospital in Karachi, Pakistan

Bushra Jamil a,b,∗, Rumina S. Hasanb, Arif R. Sarwari c, Jane Burtond,Roger Hewsond, Christopher Cleggd

a Department of Medicine, The Aga Khan University Hospital, Stadium Road, P.O. Box 3500, Karachi74800, Pakistanb Department of Pathology/Microbiology, The Aga Khan University Hospital, Karachi, Pakistanc Robert C. Byrd Health Sciences Center, West Virginia University, Morgantown, WV, USAd Novel and Dangerous Pathogens, Centre for Emergency Preparedness and Response —– Health

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Protection Agency, Porton Down, Salisbury, UK

Received 30 April 2004; received in revised form 11 March 2005; accepted 11 March 2005

KEYWORDS Summary Crimean-Congo hemorrhagic fever (CCHF) is endemic in certain rural

Crimean-Congohemorrhagic fever;Prognostic markers;

areas of Pakistan. Since the discovery of CCHF virus (CCHFV) in the country in the1960s, there have been 13 outbreaks in addition to sporadic cases. An outbreak dur-ing 2000 coincided with the movement of sacrificial animals from rural to urban areas

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Reversetranscriptase—PCR;Nucleotide sequence;Pakistan

for the festival of Eid-ul-Azha. Diagnosis was suspected in patients with fever andthrombocytopenia, and confirmed retrospectively using immunoassays and reversetranscriptase-PCR. Patients were given platelet, plasma and red cell infusions. Man-agement varied due to unfamiliarity with the condition and its treatment, lack ofavailability of diagnostic laboratory tests and limited supply of ribavirin. Inadequateantiviral treatment and late presentation probably contributed to the death of sixof the eight patients. Renal failure, disseminated intravascular coagulation and per-sistent high-grade fever were associated with mortality. The nucleotide sequenceof the small genomic RNA segment of the CCHFV isolated in this outbreak was foundto be very closely related to the CCHFV strains previously isolated in Pakistan.© 2005 Published by Elsevier Ltd on behalf of Royal Society of Tropical Medicine andHygiene.

1. Introduction

Crimean-Congo hemorrhagic fever (CCHF) is en-demic in parts of Africa, southeastern Europe,

* Corresponding author. Tel.: +92 21 4930051x4515;fax: +92 21-4934294/4932095.

E-mail addresses: bushra.jamil@aku.edu,

the Russian Federation, Central Asia and Pak-istan (Casals, 1978). The CCHF virus (CCHFV)

jamilbushra@yahoo.com, bushrajamil@hotmail.com (B. Jamil). (genus Nairovirus, family Bunyaviridae) is a small

0035-9203/$ — see front matter © 2005 Published by Elsevier Ltd on behalf of Royal Society of Tropical Medicine and Hygiene.doi:10.1016/j.trstmh.2005.03.002

578 B. Jamil et al.

enveloped virus with a genome consisting of threesegments of single-stranded RNA of negative polar-ity. The disease is transmitted through tick bite,or direct contact with viremic animals or humans.Disease manifestations range from self-limitingflu-like symptoms to overt life-threatening hem-orrhage (Leshchinskaya, 1965; Swanepoel et al.,1987).

In this study, we describe the clinical and labora-tory features in eight cases of CCHF who presentedto a tertiary care hospital in a non-endemic areaduring the year 2000 outbreak in Pakistan. We an-alyze the possible reasons for this outbreak in ur-ban Karachi during the festival of Eid-ul-Azha andthe various factors that were associated with in-creased mortality. Clinical diagnosis of CCHF in thiscase series was based on history of contact andpresence of fever and thrombocytopenia, with orwithout overt bleeding. Patient management issuesarising from lack of familiarity with the conditionand limited availability of ribavirin are discussed.Results of nucleotide sequencing of the small (S)genomic RNA segment of the CCHFV isolated fromone of the patients in this outbreak are also pre-sented to highlight the role of animal transit in dis-

for Disease Control and Prevention, Atlanta, GA,USA, where they were screened for CCHF antigens,and anti-CCHF IgG and IgM antibodies (details ofmethodology not available). Stored sera were alsosent to the Centre for Emergency Preparedness andResponse —– Health Protection Agency, Salisbury,UK, for retrospective confirmation of diagnosisusing reverse transcriptase-PCR (RT-PCR). RNA wasextracted from serum samples using the Viral RNAMini kit (Qiagen, USA) according to the manufac-turer’s protocol. The method described by Burt etal. (1998) was used to amplify a fragment of thesmall (S) RNA segment by RT-PCR. Subsequently,the primers 6486 (5′ TCTCAAAGAAACACGTGCCGCT-TACGC) and 6487 (5′ TCTCAAAGATATCGTTGCCG-CACAGCCCTT) were used to amplify the completeS RNA segment. The complete nucleotide se-quence of the S RNA segment was determinedfrom the RT—PCR products, which were amplifieddirectly from a serum specimen from patient no.3, confirming the identity of the infecting virus asCCHFV.

The SRS server at the European BioinformaticsInstitute was used to identify and recover full-length CCHFV S genomic RNA sequences. ThesewtCalsTl

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ease spread.

2. Methods

The Aga Khan University Hospital (AKUH) is a647-bed, private, tertiary care teaching hospi-tal that serves the estimated 8—12 million in-habitants of Karachi, Pakistan. The hospital alsoserves as a referral center for the nearby citiesof the Sindh Province and other areas of Pak-istan. Between September and November 2000, 29patients were admitted to AKUH with fever andthrombocytopenia. On the basis of clinical featuresand suggestive laboratory abnormalities includ-ing initial hemoconcentration followed by a dropin hemoglobin/hematocrit, leucopenia, thrombo-cytopenia and deranged coagulation, a diagnosis ofviral hemorrhagic fever (VHF) was made. Confirma-tory diagnostic tests for VHF were not available.Crimean-Congo hemorrhagic fever was strongly sus-pected in 8/29 of these patients. The cause of feverand thrombocytopenia could not be ascertained inthe rest of the cases and because of initial lackof suspicion, blood samples could not be collectedfrom these patients for retrospective confirmationof diagnosis of CCHF. These cases were thereforeexcluded from this study.

Serum samples from the suspected cases ofVHF were collected and stored at −80 ◦C. Thesesamples were subsequently sent to the Centers

ere aligned using Clustalw version 1.82, withhe inclusion of full-length S RNA sequences fromCHFV strains Baghdad-12 (Al-Tikriti et al., 1981)nd SR3 (this paper). The S RNA sequence of the re-ated Nairovirus Hazara was also included. The re-ulting alignment was analysed using the programree-Puzzle version 5.0 and the output maximumikelihood tree drawn using TreeView.

. Case reports

.1. Patients

haracteristics of the eight cases diagnosed withCHF are shown in Table 1. All were males, with ange range of 15—48 years (mean 26.4). The diag-osis was confirmed retrospectively with positiveerological tests (antigen detection, IgM and IgG)or CCHFV in 6/8 of these patients, and addition-lly by recovery of CCHFV sequences by RT—PCRn one patient (Table 1). Patients no. 1 (laboratoryechnician) and no. 6 (butcher) were from Quetta,alochistan, an area known to be endemic for CCHF.he other six patients were residents of Karachind gave no history of travel to any CCHFV-endemicrea in the few weeks prior to presentation. For pa-ient no. 2, results of confirmatory tests were notvailable. However, he was the index case for pa-ient no. 3 and remained in hospital from 13 to 16ctober 2000 with active bleeding from different

Crimean-Congo hemorrhagic fever in Pakistan 579

Table 1 Characteristics of patients with Crimean-Congo hemorrhagic fever (CCHF), Karachi, Pakistan, 2000

Patientno.

Age(years)

Occupation Residence Monthadmitted

Duration of symptomson admission (days)

CCHFV

Antigen IgM IgG RT—PCRa

1 18 Laboratorytechnician

Quetta Sept 4 − + + −

2 40 Butcherb Karachi Oct 4 Not tested3 30 Nurse Karachi Oct 3 + − − +4 16 Studentc Karachi Nov 3 + − − −5 16 Studentc Karachi Nov 5 − − − −6 28 Butcher Quetta Nov 9 + + − −7d 15 Student Karachi Nov 2 + − − −8d 48 Gas company

workercKarachi Nov 4 − + − −

a Reverse transcriptase—PCR.b Index case for patient no. 3.c No documented history of contact.d Survived.

sites. Patient no. 3, a nurse at AKUH looked afterhim and came down with high-grade fever on 19October and subsequently developed disseminatedintravascular coagulation. Patients no. 5 (includeddespite negative confirmatory tests) and no. 7 wereinhabitants of an orphanage, located in the out-skirts of Karachi. They had a history of contact withanother inhabitant with suspected VHF (outcome ofthe latter unknown). Patients no. 4 and 8 did nothave any obvious risk factors or history suggestiveof exposure to CCHF.

3.2. Clinical features

Fever and vomiting were the commonest present-ing complaints. Other clinical features, either onpresentation or during the hospital stay, were gas-trointestinal bleeding, myalgias and bleeding fromother sites of the body (Figure 1).

Fwi

The mean duration of symptoms was 4.3 days(range 2—9 days) at the time of admission. Pe-techiae and/or ecchymoses were seen in three pa-tients (patients 3, 6 and 8) and were present at thetime of admission (day 3 to 7 of onset of symptoms).

Mucosal bleeding from the respiratory (epis-taxis, hemoptysis), gastrointestinal (hematemesismelena/hematochezia) and genitourinary tracts(hematuria) manifested between 4 and 10 daysafter onset of symptoms. Six out of eight patientshad evidence of skin and/or mucosal bleeding.

Fever remained high grade (up to 40 ◦C axillary)throughout the course of illness in all six patientswho died (mean 37.9 ◦C axillary). One of these(patient 6), however, did become afebrile 48 h be-fore death. The two patients who recovered (pa-tients 7 and 8) presented with history of fever butremained afebrile throughout their hospital stay(Figure 2).

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igure 1 Frequency of clinical features of patientsith Crimean-Congo hemorrhagic fever, Karachi, Pak-stan, 2000.

igure 2 Average daily body temperatures (axillary) inatients with Crimean-Congo hemorrhagic fever, Karachi,akistan, 2000. av d: temperature in those who died; av: temperature in those who survived.

580 B. Jamil et al.

Table 2 Blood counts in patients with Crimean-Congo hemorrhagic fever (CCHF), Karachi, Pakistan, 2000

Patient no. Total leucocytecount (109/l)

Lymphocytes(%) (d)

Atypicallymphocytes (d)

Monocytes(%) (d)

Platelets (109/l)

Lowest (d) Highest (d) Lowest (d) Highest (d)

1 2.7 (11) 12.9 (5) 43 (5) − 8 (5) 13 (11) 50 (5)2 1.2 (7) 6.3 (5) 54.2 (7) − 7.2 (7) 9 (5) 18 (7)3 0.8 (7) 10.5 (14) 40.7 (8) − 19.1 (8) 12 (7) 46 (14)4 5.7 (5) 5.9 (4) 41.2 (5) + (5) 1.5 (4) 25 (4) 47 (5)5 2.2 (7) 24.2 (9) 56.4 (7) − 22 (6) 17 (8) 74 (6)6 4 (10) 7.2 (15) 32.3 (10) − 15.6 (15) 22 (11) 99 (15)7 3.7 (5) 4.8 (4) 39.5 (3) + (3) 17.5 (3) 55 (3) 184 (6)8 3.9 (13) 7.3 (17) 44.1 (16) + (15) 11.5 (13) 1 (7) 4 (11)

(d): day of illness.

3.3. Hematological abnormalities

Total leucocyte counts varied between 0.8× 109/land 24× 109/l. Leucopenia (total leucocyte count≤4× 109/l) was seen in six patients from day4 to 13 of onset (Table 2). Four patients whohad marked leucopenia (total leucocyte count<3× 109/l) died while the two patients who sur-vived had counts ≥3.7× 109/l (3.9 to 7.3× 109/l).On average, white cell counts dropped to a meanof 2.76× 109/l in those who died, while themean white cell count in those who survived was3.8× 109/l.

Lymphocytosis (≥40%) was noted during the earlyphase of infection in 6/8 patients. Atypical lympho-cytes were transiently present between days 4 and15 of onset of symptoms in the two patients whorecovered and in one of the patients who died. El-evated monocyte counts were also noted in 2/6 ofthe patients who died (mean of 12.3% in survivorsvs. 7.7% in non-survivors).

All patients were thrombocytopenic (plateletcount <150× 109 /l) from day 4 to day 19 after onsetof fever (Table 2). Platelet counts varied from 1 to

184× 109/l (normal range 150—400× 109/l). Sever-ity of thrombocytopenia did not correlate with dis-ease outcome. Of the two patients who survived,one patient (patient 8) had prolonged thrombocy-topenia, which persisted for about amonth after re-covery from infection and eventually resolved withprednisolone therapy. Bone marrow examination inthis patient revealed normal megakaryocytes. Thispatient did not have disseminated intravascular co-agulation (DIC) at any time during the course of hisillness.

Disseminated intravascular coagulation wasa feature in all the six patients who died (pa-tients 1 to 6) with prolonged prothrombin time(PT) and activated partial thromboplastin time(APTT) (Table 3). The mean value of prothrombintime in survivors was 12.5 s (control 13 s, range10.5—14.7 s) with a mean international normalizedratio (INR) of 1.01, while the mean PT in those whodied was 20.3 s (range 10.7—63 s) with a mean INRof 1.78. The range of APTT was 57 to >120 s (control29—32 s, mean 81.3 s, range 32.2 to >120 s) in thepatients who died vs. 30.1 s (range 26.3—36.8 s)in the two survivors. D-dimer (cross-linked fibrin

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Table 3 Laboratory valuesa in patients with Crimean-C

Patient no. PT/INR (s) (d) APTT (s) (d) D-dim(d)

1 35.3/3.5 (5) 80 (5) 4 (5)2 20.8/1.8 (7) >120 (5) 8 (5)3 63/7.41 (14) >120 (8—14) ND4 25.8/2.17 (4) 60.9 (4) 8 (4)5 19.9/1.67 (9) 57 (9) ND6 12.5/1.05 (10) 60.1 (10) 4 (10)7 10.5/0.87 (3) 26.3 (3) ND8 14.7/1.17(13) 36.8 (13) 0.5 (1

(d): day of illness; ND: not done. PT: prothrombin time; INR: Inttime; TB: total bilirubin; AST: aspartate aminotransferase; Aranges: APTT: control 29—32 s; D-dimer: control <0.5; TB: 0.2—a Highest value.

o hemorrhagic fever, Karachi, Pakistan, 2000

TB mg/dl(d)

AST (IU/l)(d)

ALT (IU/l)(d)

AP (IU/l)(d)

2.6 (5) ND 2061 (5) 139 (5)0.3 (5) ND 54 (5) 130 (5)2.8 (5) ND 684 (5) 121 (5)0.6 (4) ND 49 (4) 136 (4)1.3 (6) ND 22 (6) 121 (6)3.3 (10) 7536 (10) 2397 (10) 196 (10)0.5 69 (3) 34 (3) 112 (3)0.5 (13) 16 (13) 13 (13) 37 (13)

ional normalized ratio; APTT: activated partial thromboplastinlanine aminotransferase; AP: alkaline phosphatase. Normalmg/dl; AST: 8—46 IU/l; ALT: 0—55 IU/l,; AP: 28—124 IU/l.

Crimean-Congo hemorrhagic fever in Pakistan 581

Table 4 Duration of hospitalization, ribavirin therapy and disease outcome in patients with Crimean-Congo hem-orrhagic fever, Karachi, Pakistan, 2000

Patientno.

Hospitalstay (days)

Ribavirin Outcome

1 8 Not given Died day 12 of onset2 4 Not given Died day 83 9 2 g i.v. followed by 1 g 6 hourly for 4 days, 500mg 8 hourly for 1 day;

started on admission (day 4 of onset of symptoms)Died day 14

4 2 1.5 g i.v. followed by 400mg 8 hourly for 2 days; started on admission Died day 55 4 400mg i.v. 8 hourly for 4 days; started on admission Died day 96 11 400mg by mouth, 8 hourly for 9 days; started on admission Died day 197 4 720mg i.v., 6 hourly for 2 days followed by 400mg by mouth, 8

hourly for 7 days; started on admissionRecovered

8 10 Not given Recovered

derivative) was checked in five patients; meanvalue in those who died was 4.8 (control <0.5). TheD-dimer value remained within the normal range inone of the two patients who recovered. It was notchecked in the other survivor who had no evidenceof DIC. The peripheral smear appearance wassuggestive of microangiopathy in patients no. 1 andno. 4.

3.4. Hepatic and renal abnormalities

Liver function tests were deranged in three of thepatients who died; total bilirubin and aminotrans-ferases were elevated (patients 1, 3 and 6) andalkaline phosphatase was concomitantly elevatedin patient no. 6 (Table 3). One of these patients(patient 1) also had a positive hepatitis B core an-tibody (IgM and IgG), and concomitant infectionwith hepatitis B virus could have contributed tothe markedly deranged enzymes. Hepatitis serol-ogy was not done in patient no. 6. Liver functiontests were within the normal range in the otherpatients.

Serum creatinine remained normal (≤1.1mg/dl)ismpa

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3.6. Patient management

During their hospital stay, the six patients who diedall received multiple blood product transfusions,including packed cells, platelets and fresh frozenplasma. Patient no. 8 received platelet transfusionsonly and patient no. 7 did not require any bloodproducts.

The two patients who presented in the earlierpart of this outbreak of CCHF, and one patient whopresented later with mild disease, did not receiveribavirin. The drug was administered intravenouslyto four patients and orally to one patient (Table 4).The dose of ribavirin was not uniform and not in ac-cordance with previously recommended schedules(Fisher-Hoch et al., 1995), except in patient no. 3,in whom the disease was recognized promptly andribavirin was given at a loading dose of 2 gm intra-venously on the day of admission (day 4 of onset ofsymptoms). This was followed by 1 g every 6 hoursfor 4 days and 500mg every 8 hours subsequently.Treatment had to be stopped on day 5 in this patientdue to acute renal failure. Despite early institutionof treatment at an appropriate dose, the patientdied on day 12 of his illness. Of the other fivepwob

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n those who recovered, but was elevated in allix patients who died (range 1.4—7.1mg/dl; nor-al <1.2mg/dl). Four of these patients also hadroteinuria varying from + to +++ (patients 3, 4, 5nd 6).

.5. Radiological findings

he main findings in the four patients who un-erwent ultrasound examination of the abdomenere the presence of free fluid in the peritonealavity (patients 3, 5 and 6), dilated bowel loopspatient 1) and acalculous cholecystitis (patient 3).

atients who received ribavirin, only one patientho had a mild illness (patient 7) survived. Deathccurred, with or without ribavirin treatment,etween days 5 to 19 of disease onset (Table 4).

. Results of phylogenetic analysis ofCHF RNA

he nucleotide sequence of the S RNA segmentrom patient no. 3 was compared with previouslyetermined sequences in a phylogenetic analysisFigure 3). This showed that the virus was verylosely related to CCHFV strains isolated from

582 B. Jamil et al.

Figure 3 Phylogenetic analysis of Crimean-Congo hem-orrhagic fever virus (CCHFV). Sequence data base acces-sion numbers and geographic origin are indicated for eachsequence. Branch lengths are proportional to the horizon-tal distances shown; vertical separations are for clarityonly. Quartet puzzling support values (%) are shown asitalic numbers. CCHFV strains originating in Pakistan arehighlighted.

ticks and from patients in CCHF-endemic and en-zootic areas of Pakistan over the last three decades(Hewson et al., 2004).

5. Discussion

Crimean-Congo hemorrhagic fever is endemic andenzootic in parts of Pakistan (Burney et al., 1980).After the initial isolation of CCHFV from Hyalommaticks in the 1960s (Begum et al., 1970), therehave been 13 documented outbreaks in the coun-try (Altaf et al., 1998; Athar et al., 2003; Burney

et al., 1980; Casals, 1978; Fisher-Hoch et al., 1995),as well as sporadic cases from the rural enzooticareas of Balochistan. Previous outbreaks in non-endemic urban areas have been attributed to no-madic movement (Burney et al., 1980). In the year2000, a CCHF epidemic was noted in Loralai Dis-trict of Balochistan (45 reported cases) from thefirst week of September to December (unpublisheddata). This period coincided with transit of sacri-ficial animals (cattle, goats and sheep) from theNorth West Frontier Province and Balochistan toKarachi for the Islamic festival of Eid-ul-Azha. Thesacrifice, as always, took place on streets and road-side kerbs and involved close contact between ani-mals and humans. During this period, patients wereadmitted to our hospital with fever and thrombo-cytopenia, some of whom were suspected to haveCCHF. We suggest that some of the animals fromCCHF-enzootic areas may have been viremic withCCHFV and/or carried the tick on arrival in the cityand thus led to the first outbreak of CCHF in ur-ban Karachi. Viremia in animals and presence ofCCHFV-carrying ticks could not be confirmed duringthe outbreak due to non-availability of diagnostictests at that time. The tests have since becomeaCu

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vailable and epidemiological studies looking atCHFV IgG titers in cattle and humans are currentlynderway.Possible risk factors were occupational and res-

dential. Two patients (butchers) may have hadontact with viremic animals. One of these pa-ients, and one other, were resident in an en-emic/enzootic area. Factors which led to infectionn the remaining patients could not be ascertained.In our patients with CCHF, fever, vomiting and

ody aches were the commonest presenting com-laints. Fever was intermittent, as has been de-cribed previously (Swanepoel et al., 1987), andigh-grade fever throughout the course of illnessas associated with a poor outcome. This associ-tion has not been described before. Other fea-ures associated with mortality were deranged co-gulation profile as has been described previouslySwanepoel et al., 1989) and renal failure. Leucope-ia was more severe in those who died. There waso difference in the severity and duration of throm-ocytopenia between survivors and non-survivors,nd persistent thrombocytopenia was not a prog-ostic marker.Mortality in our CCHF patients was 75% compared

o the 5—30% (Casals, 1978) and 73% (Schwarz etl., 1997) reported in the literature. One of ourases had confirmed and another one had likelyosocomial transmission, which reportedly has aigher mortality, up to 80% (Suleiman et al., 1980;wanepoel et al., 1987). Late presentation and

Crimean-Congo hemorrhagic fever in Pakistan 583

inadequate antiviral treatment could also havecontributed to the high mortality in our patients.

Ribavirin has anti-CCHFV activity in vitro and inanimal models (Huggins, 1989; Tignor and Hanham,1993; Watts et al., 1989) and its use has been re-ported to improve patient outcome (Athar et al.,2003). The prophylactic (van de Wal et al., 1985)and therapeutic (van Eeden et al., 1985) efficacyof ribavirin in CCHF remains to be proven beyonddoubt, although recent reports suggest good effi-cacy (Mardani et al., 2003).

Ribavirin was not administered to all thesuspected cases because of delay in disease recog-nition in a non-endemic area and very limitedavailability of parenteral ribavirin (which was notmarketed in Pakistan at the time of the outbreak),so it was not possible to use the drug in accordancewith the recommended regimens in all the sus-pected cases. Of the five patients treated with rib-avirin, four died despite early clinical diagnosis (day3 of onset) and treatment. The early institution ofantiviral therapy did not affect the uniformly pooroutcome of CCHF patients with DIC. Given the dif-ficulties involved in obtaining parenteral ribavirinand poor health economics of the regions whereCe

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outbreak in Karachi was a consequence of spreadof CCHFV from endemic/enzootic areas of Pak-istan, to a non-endemic urban area, either throughinfected or tick-bearing cattle, goats and sheep(likely) or through other means, e.g. migrating birds(less likely). Phylogenetic studies further show thatCCHFV strains from Pakistan, the United Arab Emi-rates and Madagascar fall into the Asia 1 genogroup(Hewson et al., 2004) suggesting that the livestocktrade, and perhaps birdmigrationmay have broughtabout links between these distant locations.

In conclusion, this outbreak of VHF underscoresthe need for clinicians to recognize CCHF at an earlystage of the illness. This would help patient man-agement through early institution of ribavirin ther-apy and also ensure that appropriate precautionsare taken to prevent nosocomial spread.

Conflicts of interest statementThe authors have no conflicts of interest concerningthe cases reported in this paper.

Acknowledgements

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CHF is prevalent, administration of ribavirin to ev-ry patient with suspected VHF cannot be ensured.In view of the high mortality and the potential

or nosocomial spread (Altaf et al., 1998; Burney etl., 1980) it is important that CCHF is rapidly recog-ized and confirmed. Early recognition is challeng-ng in an area which experiences a surge in cases ofengue fever from time to time (Akram et al., 1998;ureshi et al., 1997; B. Jamil et al., unpublishedata). Additionally, history of exposure or contactith animals, infected individuals or tick bite mayot be forthcoming in many patients.Laboratory confirmation of VHF is problematichen the specific diagnostic tests are restricted tofew reference laboratories worldwide. In this out-reak, sera were sent to the Centers for Diseaseontrol and Prevention (USA) and the Centre forpplied Microbiology and Research (UK) for con-rmation, with a minimum turnaround time of 3eeks, which precluded the use of these tests foriagnosis.From an epidemiological point of view, viral

hylogenetic analysis is important to allow com-arison with other CCHF strains that have beensolated in the country over a period of time asell as with those circulating within the region.uch analysis showed that the virus isolated fromne of our patients was very closely related toCHFV strains isolated from both ticks and pa-ients in CCHF-endemic and enzootic areas of Pak-stan (Hewson et al., 2004). This suggests that the

e would like to thank Dr Amna Rehana Siddiquior her advice and Dr Stuart Nichol and the Specialathogens Branch at the Centre for Disease Controlnd Prevention, Atlanta, GA, USA for serodiagnosis.ork at Centre for Emergency Preparedness and Re-ponse on CCHFV was supported by the Departmentf Health of England and Wales and by the Well-ome Trust (project grant no. 061414). The viewsxpressed in this publication are those of the au-hors and not necessarily those of the Departmentf Health.

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