Two outbreaks of acute hemorrhagic conjunctivitis in Africa due to genotype III coxsackievirus A24 variant

Reported here are two outbreaks of acute hemorrhagic conjunctivitis that occurred in the Democratic Republic of the Congo and in Morocco in the summers of 2003 and 2004, respectively, with a large impact on public health. Virus was isolated from the conjunctival swabs of 30 Congolese and 20 Moroccan patients. Enterovirus-specific cytopathic effect was observed in all samples. None of the strains could be typed using a conventional neutralization assay with the Melnick intersecting pools; however, by sequencing the VP1 region, the viruses could be identified as coxsackie A24 variants. Phylogenetic analysis of the 3C protease region revealed that these strains were closely related to each other as well as to genotype III isolates detected in Korea in 2002, thus proving their worldwide spread. This is the first report of an epidemic of acute hemorrhagic conjunctivitis due to a coxsackievirus A24 variant in Africa since 1987 and the first ever from Morocco. An erratum to this article can be found at     

Multiple outbreaks of acute hemorrhagic conjunctivitis due to a variant of coxsackievirus A24: Guangdong, China, 2007

Acute hemorrhagic conjunctivitis (AHC) is usually caused by enterovirus 70, coxsackievirus A24(CA24v) and adenoviruses. Several outbreaks of AHC caused by a CA24v have occurred since it was imported into China in 1971. Multiple outbreaks of AHC reappeared in 10 cities of Guangdong during June to November in 2007. The epidemic began in the June, and spread extensively, with a peak in the September. A total of 31,659 cases were reported to center for disease control and prevention of Guangdong, it was estimated that the number of actual AHC was >200 thousands. Forty conjunctival swab specimens were collected from the cases diagnosed clinically with AHC. (RT)-PCR testing on these conjunctival specimens revealed the presence of an enterovirus, and this was confirmed by 16 isolates. We demonstrated the most likely etiological agent for the multiple outbreaks was a variant of coxsackievirus A24 by molecular typing using a partial VP1 sequence. Sequence comparison and phylogenetic analyses of the VP1 and 3Cpro gene regions were performed by Neighbor-joining method, the strains from different outbreaks and different geographical areas within Guangdong had no sequence divergence in 2007. The representative isolates from mainland of China including Hangzhou, Ningbo, Beijing, Yunnan, Liaoning, and Henan were analyzed in this study. Phylogenetic analysis revealed theses isolates were located in different clusters, a close phylogenetic and chronological relationship with Singaporean, South Korean and Thailand isolates had been observed. This confirms CA24v circulated in China's mainland has not evolved independently, but co-evolved with the isolates of Southeast Asia.     

An outbreak of acute hemorrhagic conjunctivitis (AHC) caused by coxsackievirus A24 variant in Pakistan

Acute hemorrhagic conjunctivitis (AHC) is a self-limiting viral infection of the eyes but having epidemic potential. In winter 2004-2005, an outbreak of acute hemorrhagic conjunctivitis (AHC) occurred in Islamabad, Pakistan. The etiological agent was confirmed as coxsackievirus A24 variant (CA24v) by virus isolation and sequencing of a part of the VP1/VP3 gene. Phylogenetic analysis in VP1 region showed that Pakistan isolates has closest matches both in Asia and Europe while in VP1/VP3 region they were more closely related to Chinese strains, suggesting their common source in Asia which is constantly evolving to cause AHC outbreaks in susceptible hosts in different parts of the world.     

Evolutionary study on the coxsackievirus A 24 variant causing acute hemorrhagic conjunctivitis by oligonucleotide mapping analysis of RNA genome

Summary The evolution of the variant of coxsackievirus A 24 (CA 24 v) which causes acute hemorrhagic conjunctivitis was explored. Using 15 isolates obtained from Southeast Asia during the period 1970–1986, the genetic distance between isolates was estimated from pairwise comparison of nucleotide changes deduced from common spots on oligonucleotide maps of the isolates. From regression analysis of the genetic distance and the time of isolation of the isolates, the evolutionary rate of CA 24 v was estimated to be 3.44×10^–4/nucleotide/month. The phylogenetic relationship of these isolates was explored using the neighbor-joining method and the modified unweighted pair group method using arithmetic averages (UPGMA). The phylogenetic tree constructed indicates that CA 24 v appeared from one focal place in July 1968±25 months, very close to the time of the first world epidemic of, then newly recognized, acute hemorrhagic conjunctivitis.     

The complete nucleotide sequence of a variant of coxsackievirus A24, an agent causing acute hemorrhagic conjunctivitis

The complete nucleotide sequence was determined for the cDNAs that represent the RNA genome of the standard strain of a variant of coxsackievirus A24, the EH24/70, one of the agents causing acute hemorrhagic conjunctivitis. The genome is 7461 nucleotide long and is polyadenylated at the 3-end terminus. Following a 750-nucleotide 5-noncoding region, there was a long open reading frame of 6642 nucleotides, which serve to encode a viral polyprotein consisting of 2214 amino acids. Comparison of the deduced amino acid sequence of the polyprotein with those of known enteroviruses allowed us to predict the possible cleavage sites. The overall structure and the organization of the RNA genome is typical for an enterovirus. Based on the similarity of the nucleotide sequence of the 5 and 3 noncoding regions, together with the amino-acid sequence of the encoded proteins, EH24/70 appeared to be closely related to polioviruses and coxsackievirus A21.The nucleotide sequence data reported in this paper will appear in the DDBJ, EMBL, and GenBank Nucleotide Sequence Databases under the accession number D90457.     

Molecular identification of coxsackievirus A24 variant,isolated from an outbreak of acute hemorrhagic conjunctivitis in Singapore in 2005

Summary An outbreak of acute hemorrhagic conjunctivitis (AHC) was reported in Singapore military camps in the year 2005. A total of 103 conjunctival swab specimens were collected from military personnel diagnosed clinically with AHC. PCR testing on these conjunctival specimens revealed the presence of an enterovirus, and this was confirmed by virus isolation. Molecular typing using a partial VP1 gene confirmed a variant of coxsackievirus A24 (CA24v) as the most likely etiological agent for the outbreak. Full-length genome sequencing was carried out on 2 selected virus strains, DSO-26SIN05 and DSO-52SIN05. Sequence comparison and phylogenetic analyses of the VP4, VP1 and 3Cpro gene regions were performed, clustering the Singapore CA24v strains with viruses originating from Asia in the post-2000 era. In addition, we report evolution rates of 4.2 × 10⁻³ and 1.0 × 10⁻³ nucleotide/year, respectively, for the VP4 capsid and 3Cpro gene regions. Our result shows a focal evolutionary point around 1965–1966, suggesting that the CA24v virus has been evolving constantly since its emergence in Singapore, nearly 40 years ago.     

Outbreak of acute hemorrhagic conjunctivitis in French Guiana and West Indies caused by coxsackievirus A24 variant: phylogenetic analysis reveals Asian import

An outbreak of acute hemorrhagic conjunctivitis occurred in French Guiana between April and July 2003, with approximately 6,000 cases in the two major cities Kourou and Cayenne. Since acute hemorrhagic conjunctivitis is not a notifiable disease in France, there was no registration of the number of cases. Therefore, these were estimated by comparing the consumption of antibiotic eye drops and ophthalmic ointments during 2002 and 2003. The outbreak rapidly spread into the Caribbean Islands, causing an outbreak in Guadeloupe in October. Viral isolates from conjunctival swabs of 16 patients were confirmed to be enterovirus by PCR directed to the 5' UTR of the genome. The isolates could not be neutralized by the Melnick intersecting pools, but were shown to be CV-A24 variant by limited sequencing within the VP1 and 3C regions of 12 strains. Phylogenetic analysis revealed that they were similar to the genotype III strains causing outbreaks in Korea 2002 and Malaysia 2003. The previous outbreak of conjunctivitis caused by CV-A24 in the Caribbean in the 1980s was also introduced from Asia, and disappeared after 3 years. This new introduction from Asia and its rapid spread into the Caribbean, where the infection disappeared after a few months, indicates that the CV-A24 variant has a different epidemiological pattern in this region compared to South East Asia, since it has not established an endemic infection. It had to be reintroduced from Asia, where it has been circulating since the 1970s.     

Molecular identification and phylogenetic study of coxsackievirus A24 variant isolated from an outbreak of acute hemorrhagic conjunctivitis in India in 2010

An outbreak of acute hemorrhagic conjunctivitis (AHC) occured in India between August and October 2010. Molecular typing by RT-PCR and sequencing of a partial VP1 region identified coxsackievirus A24 variant (CV A24v) as the serotype involved in this outbreak. Phylogenetic analysis based on the VP1 and 3C genes revealed that CV A24v strains associated with the 2010 AHC outbreak in India were genetically similar to strains from Central and South America that caused outbreaks of AHC in Cuba between 2008 and 2009 and Brazil in 2009. The result shows that the Indian strain of CV A24v may be responsible for the recent AHC outbreak in Marseille, France, in 2012.     

A simple one-step real-time RT-PCR for diagnosis of dengue virus infection

Dengue is the most important arbovirus disease in tropical and sub-tropical countries, and can be caused by infection with any of the four-dengue virus (DENV) serotypes. Infection with DENV can lead to a broad clinical spectrum, ranging from sub-clinical infection or an influenza-like disease known as dengue fever (DF) to a severe, sometimes fatal, disease characterized by hemorrhage and plasma leakage that can lead to shock, known as dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). The diagnosis of dengue is routinely accomplished by serologic assays, such as IgM and IgG ELISAs, as well as HI tests, analyzing serum samples obtained from patients with at least 7 days of symptoms onset. These tests cannot be used for diagnosis during the early symptomatic phase. In addition, antibodies against dengue are broad reactive with other flaviviruses. Therefore, a specific diagnostic method for acute DENV infection is of great interest. In that sense, the real-time RT-PCR has become an important tool that can be used for early and specific detection of dengue virus genome in human serum samples. This study describes a simple, specific, and sensitive real-time RT-PCR for early diagnosis of dengue virus infection.     

Acute haemorrhagic conjunctivitis: seroepidemiology of coxsackievirus A24 variant and enterovirus 70 in Singapore

Epidemics of acute haemorrhagic conjunctivitis caused by a variant of coxsackievirus A24 (CA24v) and enterovirus 70 (EV70) have occurred periodically in Singapore. A seroepidemiological survey conducted before the CA24v epidemic in 1985 and in the midst of the EV70 epidemic in 1983 showed a neutralising antibody prevalence (greater than or equal to 1:4) of 19.1% and 66.9% to CA24v and EV70, respectively. The seropositivity rate to both viruses was highest in children 10-14 years of age, but no sex or ethnic difference was noted. It appears from the data that an epidemic could be triggered when the herd immunity of the population falls below a critical level. There was a significant correlation in seroprevalence between CA24v and EV70 (P = 7.75 x 10(-3).     

Molecular characterization of a coxsackievirus A24 variant that caused an outbreak of acute haemorrhagic conjunctivitis in Spain, 2004

BACKGROUND: Coxsackievirus A24 variant is one of the major etiological agents involved in acute haemorrhagic conjunctivitis. STUDY DESIGN: An outbreak of acute hemorrhagic conjunctivitis occurred in the Southeast of Spain between September and November 2004. Cellular and molecular methods were used to identify and characterize the viral agent associated with the epidemic. RESULTS: Enterovirus was detected in the conjunctival swabs of 35 patients. None of the viruses isolated could be typed by conventional neutralization assays; however, amplification and sequencing of the 3'-end VP1 region of 19 of the samples identified coxsackievirus A24 variant as the serotype causing the outbreak. Phylogenetic analysis of the 5'-half VP1 region of the genome revealed that Spanish sequences, like other strains isolated during outbreaks of hemorrhagic conjunctivitis in American and African countries in 2003 and 2004, were closely related to the isolates detected in Korea (2002), thus proving their worldwide spread. CONCLUSIONS: This is the first report of an epidemic of acute hemorrhagic conjunctivitis due to a coxsackievirus A24 variant in Spain. Molecular typing in combination with phylogenetic analysis is useful to study the enterovirus epidemiology associated with epidemics.     

Rapid detection, serotyping and quantitation of dengue viruses by TaqMan real-time one-step RT-PCR

The use of the polymerase chain reaction (PCR) in molecular diagnosis is now accepted worldwide and has become an essential tool in the research laboratory. In the laboratory, a rapid detection, serotyping and quantitation, one-step real-time RT-PCR assay was developed for dengue virus using TaqMan probes. In this assay, a set of forward and reverse primers were designed targeting the serotype conserved region at the NS5 gene, at the same time flanking a variable region for all four serotypes which were used to design the serotype-specific TaqMan probes. This multiplex one-step RT-PCR assay was evaluated using 376 samples collected during the year 2003. These groups included RNA from prototype dengue virus (1-4), RNA from acute serum from which dengue virus was isolated, RNA from tissue culture supernatants of dengue virus isolated, RNA from seronegative acute samples (which were culture and IgM negative) and RNA from samples of dengue IgM positive sera. The specificity of this assay was also evaluated using a panel of sera which were positive for other common tropical disease agents including herpes simplex virus, cytomegalovirus, measles virus, varicella-zoster virus, rubella virus, mumps virus, WWF, West Nile virus, Japanese encephalitis virus, S. typhi, Legionella, Leptospira, Chlamydia, and Mycoplasma. The sensitivity, specificity and real-time PCR efficiency of this assay were 89.54%, 100% and 91.5%, respectively.     

Novel one-step real-time RT-PCR assay for rapid and specific diagnosis of Crimean-Congo hemorrhagic fever encountered in the Balkans

Early and accurate diagnosis of Crimean-Congo hemorrhagic fever (CCHF) is essential for the treatment and outcome of the disease and prevention of its further transmission. Molecular-based diagnostic assays now serve as the front-line tool in the diagnosis of CCHF. However, the development of real-time RT-PCR assay for the detection of Crimean-Congo hemorrhagic fever virus (CCHFV) has been hampered by a virus strain variation. The development of a one-step real-time RT-PCR assay for the detection of CCHFV is described herein. The technique is based on the fluorescence resonance energy transfer probe technology employing the endonuclease activity of Taq polymerase enzyme. The assay was designed to detect specifically the strains from a phylogenetic cluster of CCHFV which encompasses the known CCHFV strains circulating in the Balkan region. The detection system was tested using CCHFV strain Kosovo Hoti, clinical serum samples and ticks. The real-time assay described is rapid, specific and sensitive. Since the Balkan peninsula is also an endemic region for hemorrhagic fever with renal syndrome (HFRS), this method is suggested as convenient for early differential diagnosis of suspected viral hemorrhagic fever patients.     

Outbreak of acute hemorrhagic conjunctivitis in Maharashtra and Gujarat states of India, caused by Coxsackie virus A-24 variant

Acute hemorrhagic conjunctivitis is associated with enteroviruses. Among these, Coxsackie A-24 variant (CA-24) and Enterovirus-70 (EV-70) are known to cause epidemics and pandemics. An outbreak of acute hemorrhagic conjunctivitis occurred in August-September 2003 in Maharashtra and Gujarat states of India. The present investigation was carried out to determine the viral etiological agent associated with the epidemic. Virus isolates were obtained from 11 eye swabs of conjunctivitis patients using HeLa/ Hep-2 cell lines. The isolates were characterized by serological and mouse pathogenecity tests, RT-PCR using enterovirus common primers (VP4-VP2), CA-24 specific primers (3C-proteinase region), EV-70 primers (VP-3) followed by sequencing, and phylogenetic analysis. The virus was characterized as a Coxsackie A-24 variant (CA-24v) and none of the isolates were found to be positive for EV-70. Sequencing of the PCR products derived from all the 11 isolates revealed 98.4% (SE 0.20) nucleotide identity within the Indian strains and 98.6% (0.50) and 94.4% (0.30) nucleotide identity respectively with the West Indies and Asian strains reported worldwide. The findings suggest that the outbreak of acute hemorrhagic conjunctivitis that occurred in Maharashtra and Gujarat states of India during August-September 2003 was caused by the Coxsackie A-24 variant (CA-24v).     

柯萨奇病毒A24变种的血清抗体检测

目的　观察急性出血性结膜炎（ＡＨＣ）流行后人群中柯萨奇病毒Ａ２４ 变种（ＣＡ２４ ｖ） 感染情况（或状态）。方法　采用中和试验测定人群中血清抗体：血清作１∶１０ 稀释,病毒用１００ＴＣＩＤ５０ 在微量培养板上进行。ＣＡ２４ ｖ 病毒与血清等量混合,３７℃结合１ 小时,接种细胞,观察细胞病变。结果　ＡＨＣ流行后城市居民ＣＡ２４ ｖ 血清中和抗体阳性率为４９－６７％ ,其中１９～２５ 岁年龄组约占６９－４９％ ,统计学上有显著性（ Ｐ＜ ０－０１） 。结论　ＡＨＣ流行后人群的抗体水平,反映人群受ＣＡ２４ ｖ 感染状态、抗体阳性率与年龄有关,可能反映不同年龄人群参加社会活动多少所致