Chikungunya Disease Outbreak, Reunion Island

During 2005, the monthly CDR remained within expected range of statistical variation. From January through April 2006, respectively, monthly CDRs were 7.1%, 34.4%, 25.2%, and 8.3% higher than expected rates (p 75 years of age.

During 2005, the monthly CDR remained within expected range of statistical variation. From January through April 2006, respectively, monthly CDRs were 7.1%, 34.4%, 25.2%, and 8.3% higher than expected rates (p<0.01 for February and March). This corresponded to 226 excess deaths reported by the 13 offices participating in the study and 260 excess deaths when data were extrapolated to the entire population of the island (an increase of 18.4%) (Figure). The 260 excess deaths is a crude figure that includes potentially all causes of death. This figure leads to a rough estimate of the case-fatality rate for chikungunya disease of ≈1/1,000 cases. Excess deaths were observed mainly in persons >75 years of age.
CDRs began to exceed the expected range during the last week of January 2006 and remained elevated until the end of the study period. This situation closely matched the kinetics of the epidemic curve of chikungunya disease. CDR is a stable variable in time for a defined population. Only a massive phenomenon can have an effect on it, and no other abnormal health event affected the island at this time. Thus, the outbreak of chikungunya disease was likely responsible for most of the excess deaths observed in Reunion during the first 4 months of 2006.
Deaths associated with chikungunya disease have been rarely reported. This outbreak in Reunion is the first with such a high incidence in a setting where real-time death reporting is a standard procedure. In such settings, CDR monitoring should be considered syndromic surveillance and should be implemented when an abnormal health phenomenon affects large populations. Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 12, No. 12, December 2006

Assay to Detect H5N1 Oseltamivir Resistance
To the Editor: Oseltamivir is a neuraminidase inhibitor approved for treatment and prevention of influenza virus infection. Oseltamivir resistance caused by a single amino acid substitution from histidine (H) to tyrosine (Y) at position 274 of the neuraminidase active site has been reported in persons infected both experimentally and naturally with influenza A virus subtype H5N1 (1,2). Evidence suggests that using lower doses of oseltamivir or shorter-than-recommended treatment periods can contribute to emergence of viral resistance (1,3) Currently, oseltamivir is being used in several countries that may be affected by epidemics of H5N1. Therefore, monitoring for oseltamivir-resistant strains of H5N1 during oseltamivir administration is essential for outbreak management and prevention.
Although real-time PCR or pyrosequencing is more rapid than high-throughput assays for mutation detection (4,5), the conventional PCR technique can be applied to detect drug-resistant mutation (6) in areas lacking real-time PCR or pyrosequencing capabilities. Therefore, to discriminate between oseltamivirsensitive and oseltamivir-resistant strains, we developed a simple method, based on PCR, which takes advantage of the H274Y substitution. The forward primer was designed from the conserved region common to both wild-type and mutant strains; the reverse primers were designed specifically for wild-type and mutant strains, respectively, derived from the 3′ terminal base of each primer. The primers consisted of a forward primer N1f (nt 517-534: 5′-GGGGCTGTG-GCTGTATTG-3′) and reverse primer H274r (nt 759-784: 5′-GGATAA-CAGGAGCAYTCCTCATAGTG -3′) for wild-type strain detection or Y274r (nt 759-784: 5′-GGATAACA-GGAGCAYTCCTCATAGTA-3′) for mutant strain detection. (Note: Underlined letters represent differences in nucleotides between plus vs. minus primers.) Both strains yielded products of ≈267 bp; hence, the assay consisted of 2 separate reactions for detecting wild-type and mutant strains, respectively.
To optimize the assay, we performed PCR-based H274Y mutagenesis of the N1 fragment of the H5N1 virus (primers on request). The resulting mutagenic and wild-type products were cloned into the pGEM-T Easy Vectors (Promega, Madison, WI, USA), confirmed by direct sequenc-ing, and then used as positive controls. Preliminary results showed that the wild-type primer was specific for the oseltamivir-sensitive strain, whereas the mutant primer can be used to detect the oseltamivir-resistant strain exclusively because no significant cross-amplification had been observed.
To establish sensitivity, serial 10fold dilutions of the standard N1 plasmids (wild-type and mutant) ranging from 10 9 to 10 1 copies/µL were used as a template. The threshold concentrations for detection of wild-types and mutants were 10 3 copies/µL. To provide semiquantitative data to detect subpopulations of the resistant variants, the 2 control plasmids were mixed at wild-type:variant ratios of 10 8 :10 2 , 10 7 :10 3 , 10 6 :10 4 , 10 5 :10 5 , 10 4 : 10 6 , 10 3 :10 7 , and 10 2 :10 8 . The result showed that the density of the expected bands depended on the amount of DNA templates ( Figure B). However, the mixing experiments indicated that the predominant mixtures of wildtype:resistant variant were 80:20, which is the lowest ratio of resistant variants that the assay can reliably detect (data not shown). To assess specificity, human DNA and viral cDNA extracted from other subtypes of influenza A virus (N2-N9) were subjected to this assay. No cross-reaction occurred with human DNA or other subtypes of influenza A virus.
We further validated the assay by testing 3 specimens from hosts treated with oseltamivir and 17 specimens from untreated hosts; infection with H5N1 was detected by using multiplex real-time PCR (7). The specimens from oseltamivir recipients were isolated from a Vietnamese patient on the third day after he received a prophylactic dose (1) and from 2 tigers (CU-T7 and KU-11) (8). The specimens from untreated hosts were isolated from the plasma of an H5N1-infected human (9) and from virus-containing allantoic fluid isolated from infected chickens, ducks,