Zika virus infection, Cambodia, 2010.
Article Type: Letter to the editor
Subject: Polymerase chain reaction (Usage)
Authors: Heang, Vireak
Yasuda, Chadwick Y.
Sovann, Ly
Haddow, Andrew D.
da Rosa, Amelia P. Travassos
Tesh, Robert B.
Kasper, Matthew R.
Pub Date: 02/01/2012
Publication: Name: Emerging Infectious Diseases Publisher: U.S. National Center for Infectious Diseases Audience: Academic; Professional Format: Magazine/Journal Subject: Health Copyright: COPYRIGHT 2012 U.S. National Center for Infectious Diseases ISSN: 1080-6040
Issue: Date: Feb, 2012 Source Volume: 18 Source Issue: 2
Topic: Event Code: 310 Science & research
Geographic: Geographic Scope: Cambodia Geographic Code: 9CAMB Cambodia
Accession Number: 281175428
Full Text: To the Editor: Zika virus (ZIKV), a member of the family Flaviviridae, genus Flavivirus, was first isolated from the blood of a sentinel rhesus monkey from the Zika Forest ofUganda in 1948 (1). Since that time, serologic studies and virus isolations have demonstrated that the virus has a wide geographic distribution, including eastern and western Africa; the Indian subcontinent; Southeast Asia; and most recently, Micronesia (2-5). The virus is transmitted primarily through the bite of infected mosquitoes and most likely is maintained in a zoonotic cycle involving nonhuman primates (1), although recent evidence suggests the possibility of occasional sexual transmission in humans (4). Few case reports have described the clinical characteristics of ZIKV infection in humans. Most reports describe a self-limiting febrile illness that could easily be mistaken for another arboviral infection, such as dengue or chikungunya fever. We report a confirmed case of ZIKV infection in Cambodia.

Since 2006, the US Naval Medical Research Unit No. 2 (NAMRU-2) has conducted surveillance for acute fever to determine causes of the infection among patients who seek health care at local clinics in Cambodia. Patients were enrolled by the health clinic physician after they gave informed consent in accordance with an institutional review board protocol approved by NAMRU-2 and the National Ethics Committee for Human Research of Cambodia. At enrollment, the physician administered a questionnaire and collected specimens (blood and throat swabs). All items were transported to the NAMRU-2 laboratory in Phnom Penh, where testing was conducted for a variety of viral, bacterial, and parasitic pathogens. In August 2010, a blood specimen was collected from a 3-year-old boy at a health clinic in Kampong Speu Province, Cambodia. The child's reported clinical symptoms included 4 days of fever and sore throat and cough and a headache for 3 days. A maculopapular rash was not observed, and the boy was not hospitalized. The clinic staff conducted a follow-up interview and reported that the patient recovered fully.

ZIKV infection was confirmed in this patient by using PCR, sequencing, and serology and through virus isolation. ELISA for chikungunya and dengue virus IgM and IgG antibodies on acute- and convalescent-phase serum was negative. A universal flavivirus real-time PCR screen that targets the nonstructural (NS) 5 gene (6) determined that the patient's serum was positive for flavivirus RNA, but subsequent species-specific PCR ruled out 2 other flaviviruses that are highly endemic to the region (dengue and Japanese encephalitis viruses) (7-9). This result was the first nondengue, non-Japanese encephalitis virus flavivirus detected after samples from -10,000 enrolled patients were tested. Nucleic acid sequencing of the amplicon isolated by gel purification produced a 100bp fragment with 100% sequence identity to ZIKV (nucleotide position 8,969 of the NS5 gene of the isolate GenBank accession no. EU545988). ZIKV infection subsequently was serologically confirmed by hemagglutination-inhibition tests on paired serum samples. The patient's acute-phase sample was negative, but a convalescent-phase sample gave a positive reaction with ZIKV antigen to a serum dilution of 1:320 and was negative to antigens for the 4 dengue serotypes and yellow fever and West Nile viruses. These results demonstrate that the patient had a clear monotypic flavivirus immune response with seroconversion against ZIKV, indicating a recent primary infection.

The most common signs and symptoms reported in confirmed ZIKV infections are fever, headache, malaise, maculopapular rash, fatigue or myalgia, and arthritis and arthralgia (Table). In addition to fever and headache, the patient in this study had a sore throat and cough. Because of the patient's age, additional information about symptoms was difficult to obtain.

The clinical characteristics exhibited by this case-patient are similar to those of shown in a small cluster of ZIKV infections described in Indonesia during 1977-1978 in which maculopapular rash was not observed (5). Maculopapular rash was reported as a common sign in case-patients from the recent Yap Island outbreak (3), as well as in case reports from Uganda (2), Senegal, and the United States (4), A case report of laboratory-acquired ZIKV infection also noted the lack of maculopapular rash (10).

The clinical features of ZIKV infection are similar to those of dengue virus and chikungunya virus infections, and both arboviruses are found in Southeast Asia. In this region, laboratory-based confirmation is essential. The extent of ZIKV infections in Cambodia is unknown; further studies are needed to clarify the prevalence and geographic distribution of ZIKV infection in the country.

Acknowledgments

We thank the enrolling health center staff at Kampong Speu Province and the NAMRU-2 staff who contributed to the execution of this study and the follow-up investigation.

This study was funded by the US Department of Defense Global Emerging Infections Surveillance and Response System, a division of the Armed Forces Health Surveillance Center.

Vireak Heang, Chadwick Y. Yasuda, Ly Sovann, Andrew D. Haddow, Amelia P Travassos da Rosa, Robert B. Tesh, and Matthew R. Kasper [1]

Author affiliations: US Naval Medical Research Unit No. 2, Phnom Penh, Cambodia (V. Heang, C.Y. Yasuda); Ministry of Health, Phnom Penh (L. Sovann); University of Texas Medical Branch, Galveston, Texas, USA (A.D. Haddow, A.P. Travassos da Rosa, R.B. Tesh); and US Naval Medical Research Unit No. 2, Jakarta, Indonesia (M.R. Kasper)

DOI: http://dx.doi.org/10.3201/eid1802.111224

References

(1.) Dick GW, Kitchen SF, Haddow AJ. Zika virus. I. Isolations and serological specificity. Trans R Soc Trop Med Hyg. 1952;46:509-20. http://dx.doi.org/ 10.1016/0035-9203(52)90042-4

(2.) Simpson DI. Zika virus infection in man. Trans R SocTrop Med Hyg. 1964;58:335-8. http://dx.doi.org/10.1016/0035-9203(64) 90201-9

(3.) Duffy MR, Chen TH, Hancock WT, Powers AM, Kool JL, Lanciotti RS, et al. Zika virus outbreak on Yap Island, Federated States of Micronesia. N Engl J Med. 2009;360:2536-43. http://dx.doi. org/10.1056/NEJMoa0805715

(4.) Foy BD, Kobylinski KC, Chilson Foy JL, Blitvich BJ, Travassos da Rosa A, Haddow AD, et al. Probable non-vector-borne transmission of Zika virus, Colorado, USA. Emerg Infect Dis. 2011;17:880-2.

(5.) Olson JG, Ksiazek TG. Suhandiman, Triwibowo. Zika virus, a cause of fever in Central Java, Indonesia. Trans R Soc Trop Med Hyg. 1981;75:389-93. http://dx.doi. org/10.1016/0035-9203(81)90100-0

(6.) Moureau G, Temmam S, Gonzalez JP, Charrel RN, Grard G, de Lamballerie X. A real-time RT-PCR method for the universal detection and identification of flaviviruses. Vector Borne Zoonotic Dis. 2007;7:467-77. http://dx.doi.org/10.1089/ vbz.2007.0206

(7.) Santhosh SR, Parida MM, Dash PK, Pa teriya A, Pattnaik B, Pradhan HK, et al. Development and evaluation of SYBR Green I-based one-step real-time RTPCR assay for detection and quantitation of Japanese encephalitis virus. J Virol Methods. 2007;143:73-80. http://dx.doi. org/10.1016/j.jviromet.2007.02.011

(8.) Reynes JM, Ong S, Mey C, Ngan C, Hoyer S, Sall AA. Improved molecular detection of dengue virus serotype 1 variants. J Clin Microbiol. 2003;41:3864-7. http://dx.doi. org/10.1128/JCM.41.8.3864-3867.2003

(9.) Lanciotti RS, Calisher CH, Gubler DJ, Chang GJ, Vorndam AV. Rapid detection and typing of dengue viruses from clinical samples by using reverse transcriptasepolymerase chain reaction. J Clin Micro biol. 1992;30:545-51.

(10.) Filipe AR, Martins CM, Rocha H. Laboratory infection with Zika virus after vaccination against yellow fever. Arch Gesamte Virusforsch. 1973;43:315-9. http://dx.doi. org/10.1007/BF01556147

[1] Current affiliation: US Naval Medical Research Unit No. 6, Lima, Peru.

Address for correspondence: Matthew R. Kasper, US Embassy Lima, NAMRU-6, Unit 3230, Box 46, DPO AA 34031, USA; email: matthew.kasper@med.navy.mil

The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the Centers for Disease Control and Prevention or the institutions with which the authors are affiliated.
Table. Reported or observed clinical signs and symptoms in
persons with Zika virus infection, 1962-2010 Country, year
of infection origin, * no. (%) patients

                                         Laboratory    Indonesia,
                             Uganda,      acquired,    1977-1978,
Sign or symptom            1962, n = 1   1973, n = 1     n = 7

Fever                        1 (100)       1 (100)      7 (100)
Headache                     1 (100)
Malaise                      1 (100)                     5 (71)
Maculopapular rash           1 (100)
Fatigue or myalgia           1 (100)       1 (100)       1 (14)
Arthritis and arthralgia                                 1 (14)
Chills                                     1 (100)       2 (29)
Dizziness                                                5 (71)
Joint swelling or edema
Stomachache                                              6 (86)
Retro-orbital pain                         1 (100)
Conjunctivitis                                           1 (14)
Anorexia                                                 4 (57)
Photophobia
Vomiting                                                 1 (14)
Diarrhea                                                 3 (43)
Constipation                                             3 (43)
Sore throat
Cough
Aphthous ulcer
Hypotension                                              2 (29)
Hematuria                                                1 (14)
Prostatitis
Hematospermia
Sweating                                   1 (100)
Lightheadedness
                                          Senegal/United
                           Micronesia,    States, 2009,     Cambodia,
Sign or symptom            2007, n = 28       n = 3        2010, n = 1

Fever                        20 (65)                         1 (100)
Headache                     14 (45)         3 (100)         1 (100)
Malaise                                      3 (100)
Maculopapular rash           28 (100)        3 (100)
Fatigue or myalgia           14 (45)          1 (33)
Arthritis and arthralgia     20 (65)         3 (100)
Chills                                        2 (67)
Dizziness
Joint swelling or edema       6 (19)          2 (67)
Stomachache
Retro-orbital pain           12 (39)
Conjunctivitis               17 (55)          1 (33)
Anorexia
Photophobia                                   1 (33)
Vomiting                      3 (10)
Diarrhea
Constipation
Sore throat                                                  1 (100)
Cough                                                        1 (100)
Aphthous ulcer                                2 (67)
Hypotension
Hematuria
Prostatitis                                   1 (33)
Hematospermia                                 1 (33)
Sweating
Lightheadedness                               1 (33)

* References: Uganda (2), laboratory/acquired (10), Indonesia
(5), Micronesia (9), Senegal/United States (4). Blank cells
indicate no reported information.
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