In 2001, Myanmar (Burma) had its largest outbreak of dengue���15,361 reported cases of dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS), including 192 deaths. That year, 95% of dengue viruses isolated from patients were serotype 1 viruses belonging to two lineages that had diverged from an earlier, now extinct, lineage sometime before 1998. The ratio of DHF to DSS cases in 2001 was not significantly different from that in 2000, when 1,816 cases of DHF/DSS were reported and dengue 1 also was the most frequently isolated serotype. However, the 2001 ratio was significantly higher than that in 1998 (also an outbreak year) and in 1999, when all four serotypes were detected and serotypes 1, 2, and 3 were recovered in similar numbers. The large number of clinical cases in 2001 may have been due, in part, to a preponderance of infec- tions with dengue 1 viruses. Dvivirusisof engue a disease caused by four serotypes of a fla- the same name (1). Infection with these viruses may be inapparent, or it may result in disease vary- ing in severity from a mild influenza-like illness to hemor- rhagic fever and hypovolemic shock, which may be fatal if untreated (1). Dengue is an important source of illness and death in tropical nations, particularly in Southeast Asia and Central and South America (2). In 1998, a pandemic of dengue resulted in 1.2 million cases of dengue hemorrhag- ic fever (DHF) in 56 countries (3). In many countries in Asia where this disease is endemic, outbreaks occur in cycles of 3 to 5 years due, perhaps, to enhanced infection with one serotype caused by cross-reactive antibody pro- duced in response to an earlier infection with a second serotype (4), rather than to climatic effects (5). Furthermore, the incidence of disease and its severity vary between primary and secondary infections and between infections with different dengue virus serotypes (6���8). All four dengue virus serotypes circulate in countries in Southeast Asia from Myanmar to Indonesia (9���13), and no outbreaks caused by single virus serotypes, as have been seen in dengue non-endemic areas such as Cuba or Australia (14,15), have recently been reported. Phylogenetic studies have shown regular extinction of strains of dengue viruses in single locations and emergence of new strains (16,17), and it has been suggested that the appearance of more fit or more pathogenic viruses may occur as a result of immunologic selection during periods of intense transmission during outbreaks (18). While weak selective pressure on the envelope (E) protein gene of some dengue viruses is evident (19), the most extensive changes in virus genotypes appear to be due to recombina- tion or to possible genetic bottlenecks (16,17,20). Given these observations, and the finite pool of hosts in most locations (humans and selected species of Aedes mosqui- toes), perhaps it is surprising that greater competition between the four serologically related serotypes of dengue virus has not been observed, e.g., the complete exclusion of two or three serotypes from an ecologic niche. Patients and Methods Serology Acute- and, when possible, convalescent-phase serum samples were obtained from patients admitted to the Yangon Children���s Hospital with a clinical diagnosis of DHF (1). A patient with a confirmed case of dengue fever was one who met any of the following criteria: 1) paired sera showed a fourfold or greater rise in hemagglutination inhibiting (HI) antibody titer against dengue virus (21) 2) a convalescent-phase serum sample produced an immunoglobulin (Ig) G reaction (titer equivalent to an anti-dengue virus HI titer of 2,560), an IgM reaction in a commercial ���rapid��� dengue test (22), or both or 3) dengue Myanmar Dengue Outbreak Associated with Displacement of Serotypes 2, 3, and 4 by Dengue 1 Hlaing Myat Thu,*��� Kym Lowry,* Thein Thein Myint,��� Than Nu Shwe,��� Aye Maung Han,��� Kyu Kyu Khin,�� Kyaw Zin Thant,��� Soe Thein,��� and John Aaskov* Emerging Infectious Diseases ��� www.cdc.gov/eid ��� Vol. 10, No. 4, April 2004 593 *Queensland University of Technology, Brisbane, Australia ���Department of Medical Research, Yangon, Myanmar ���Yangon Children���s Hospital, Yangon, Myanmar and ��Mawlamyaing General Hospital, Mawlamyaing, Myanmar

virus was recovered by culturing 140 ��L of acute-phase serum on 25-cm2 monolayers of C6���36 A. albopictus cells for 7 days. The serotype of the virus was determined by performing indirect immunofluorescence (23) on cells from the cultures of C6���36 cells used for virus isolation with flavivirus-, dengue-, or serotype-specific monoclonal antibodies (23,24). Phylogenetic Analyses RNA was extracted from virus isolates with a commer- cial kit (Qiagen, Hilden, Germany) according to the man- ufacturer���s instructions, transcribed to cDNA, and amplified by polymerase chain reaction as described previ- ously (24). The cDNA was purified and then sequenced on an automated sequencer (Applied Biosystems, Inc., Foster City, CA) (24). Nucleotide sequences were edited, com- pared, and analyzed with software (EclustalW, Ednapars, Ednadist, Ekitsch) from the Australian Genome Information Service (available from: http://www.angis. org.au). Additional nucleotide sequences used in the phy- logenetic analyses are listed as country, year of isolation, dengue virus serotype, and GenBank accession number, i.e., (Sin[gapore]90D1, M87512 Abid[jan]99D1, AF298807 Mal[aysia]72D1-12, AF231721 Thai64D1, AF180817 Thai58D1, D10513 Phil[ippines]D1, D00503 Nauru74 D1, U88535 Mex[ico]83D1, D00504 Jam[aica]77D1, D00501 Japan43D1M, AB074760 Haw[aii]44D1, X76219 Lao96D1, AB003090 China 80D1, AF350498 Camb[odia]98D1, AF309641). Results Dengue is endemic in Myanmar. Outbreaks have occurred in 3- to 5-year cycles of increasing magnitude since the first recorded outbreak in the country in 1970 (Table 1). The outbreak in 2001 (15,361 cases of DHF/dengue shock syndrome [DSS]) was the largest on record. In 1998 and 1999, all four dengue virus serotypes (DENV-1���4) were recovered from patients in the Yangon Children���s Hospital DENV-1, -2, and -3 were recovered in approximately similar ratios each year (Table 2). In 2000 and 2001, no DENV-4 was recovered, and DENV-1 was recovered more frequently than any other serotype. In 2001, 95% of isolates were DENV-1. No significant differ- ence (p 0.05, chi-square test) was found in the rate of iso- lation of dengue viruses from seronegative serum samples for each of these years. Accompanying the change in the relative proportions of dengue virus serotypes recovered from patients in the Yangon Children���s Hospital was a significant change in the relative proportion of clinically diagnosed DHF and DSS cases (1998: 3,194 DHF, 1,402 DSS 1999: 1,741 DHF 601, DSS 2000: 896 DHF, 224 DSS 2001: 4,511 DHF, 1,105 DSS), i.e., DSS occurred in a smaller proportion of patients in 2000 and 2001 than in 1999 or 1998 (p 0.01, chi-square test). Hemorrhagic signs and symptoms devel- oped in most of the dengue fever patients in 2001 such patients were distinguished from DHF patients only on the basis that their platelet levels were 100,000/mm3. Of the patients with a laboratory-confirmed dengue infection and sufficient clinical and laboratory detail to confirm the grade of infection (990 patients), almost half (455) had a primary infection. However, DSS was more prevalent in patients with secondary infections (112/535) than in those with a primary infection (43/455). The medi- an age of patients with primary infections (5 years) was not significantly different (p 0.05, Wilcoxon rank sum) from those with a secondary infection (6 years). Thirty-nine of the primary infections occurred in children 1 year of age shock developed in 7 of these children. Of the remainder, 17 had DF, 12 had DHF grade I, and 3 had DHF grade II). Virus (dengue 1) was recovered from the acute-phase serum of three of these seven DSS patients. Two patients 1 year of age had a secondary infection dengue fever developed in both. Phylogenetic analyses of the nucleotide sequences of the E protein gene of the only pre-1998 DENV-1 available from Myanmar along with 3 of the 9 isolates from 1998, both 1999 isolates, 5 of the 6 isolates from 2000, and 8 of the 115 isolates from 2001 (including an isolate recovered from a single female A. aegypti mosquito [My01D1m193] collected in the home of a dengue patient [My01D141500]) suggested that two new strains of DENV-1 had appeared some time before 1998, i.e., all three clades of Myanmar DENV-1 viruses have 1998 viruses in them (Figure 1). The clade containing the 1996 594 Emerging Infectious Diseases ��� www.cdc.gov/eid ��� Vol. 10, No. 4, April 2004 RESEARCH Table 1. Annual dengue hemorrhagic fever cases, Myanmar Y Cases Y Cases Y Cases Y Cases 1970 1,654 1978 2,029 1986 2,114 1994 11,647 1971 691 1979 4,685 1987 7,331 1995 2,218 1972 1,013 1980 2,026 1988 1,178 1996 1,854 1973 349 1981 1,524 1989 1,196 1997 4,006 1974 2,477 1982 1,706 1990 6,318 1998 12,918 1975 6,750 1983 2,756 1991 6,770 1999 5,753 1976 3,153 1984 2,273 1992 1,685 2000 1,816 1977 5,364 1985 2,666 1993 1,979 2001 15,361