Detection of Dobrava hantavirus RNA in Apodemus mice in Bulgaria.

Several Hantaviruses cause hemorrhagic fever with renal syndrome (HFRS) in Europe: Dobrava-Belgrade virus (DOBV), Puumala, Saaremaa, Sochi, and Seoul virus. Although HFRS is endemic in Bulgaria, genome sequences of hantaviruses have never been detected in wild rodents. To identify rodent reservoirs, a total of 691 rodents from three endemic regions were trapped in 2011-2012 and screened by TaqMan RT-PCR for detection of hantaviral genomic RNA. Partial small (S) and/or large (L)-segment sequences were recovered from six Apodemus mice: five of the species A. flavicollis and one A. agrarius. Phylogenetic analysis revealed that all recovered sequences belonged to DOBV. On the phylogenetic trees, the novel Bulgarian hantavirus sequences clustered together with sequences of established previously DOBV variants recovered from Bulgarian HFRS patients and also with variants found in wild rodents trapped in Slovenia, Greece, and Slovakia. One of the novel Bulgarian DOBV S-sequences from A. agrarius was related closely to DOBV sequences recovered from A. flavicollis, suggesting a spillover of DOBV from its natural host to A. agrarius mice. The results of this study confirmed the circulation of DOBV in wild rodents in Bulgaria. The complexity of the epidemiological situation in the Balkans requires further studies of hantaviruses in rodent hosts and human HFRS cases.

Real-time reverse transcription-PCR assay panel for Middle East respiratory syndrome coronavirus.

A new human coronavirus (CoV), subsequently named Middle East respiratory syndrome (MERS)-CoV, was first reported in Saudi Arabia in September 2012. In response, we developed two real-time reverse transcription-PCR (rRT-PCR) assays targeting the MERS-CoV nucleocapsid (N) gene and evaluated these assays as a panel with a previously published assay targeting the region upstream of the MERS-CoV envelope gene (upE) for the detection and confirmation of MERS-CoV infection. All assays detected ≤10 copies/reaction of quantified RNA transcripts, with a linear dynamic range of 8 log units and 1.3 × 10(-3) 50% tissue culture infective doses (TCID50)/ml of cultured MERS-CoV per reaction. All assays performed comparably with respiratory, serum, and stool specimens spiked with cultured virus. No false-positive amplifications were obtained with other human coronaviruses or common respiratory viral pathogens or with 336 diverse clinical specimens from non-MERS-CoV cases; specimens from two confirmed MERS-CoV cases were positive with all assay signatures. In June 2012, the U.S. Food and Drug Administration authorized emergency use of the rRT-PCR assay panel as an in vitro diagnostic test for MERS-CoV. A kit consisting of the three assay signatures and a positive control was assembled and distributed to public health laboratories in the United States and internationally to support MERS-CoV surveillance and public health responses.

Seroprevalance of Crimean-Congo haemorrhagic fever in Bulgarian livestock.

Crimean-Congo haemorrhagic fever (CCHF) is a tick-borne zoonotic disease. Over the past decade, CCHF cases in humans have emerged in Turkey and reemerged in the Balkan countries, Ukraine and Tajikistan. Occupational contact with infected livestock has been recognized as a common cause of the disease. A cross-sectional seroprevalence study in livestock was conducted in farming communities of an endemic area in Bulgaria, southeastern Europe. Overall, 72% of the tested animals were positive for IgG antibodies to CCHF virus. By the time the animals were one-year old almost 50% had serologic evidence of CCHF infection, and by two years already 80% of them had been infected. The data obtained in this study reflect current situation of CCHF virus infection among livestock in Bulgaria. The results showed active CCHF virus circulation that poses risk for humans to be infected during contacts with animals and requires public health awareness.

Microevolution of highly pathogenic avian influenza A(H5N1) viruses isolated from humans, Egypt, 2007-2011.

We analyzed highly pathogenic avian influenza A(H5N1) viruses isolated from humans infected in Egypt during 2007-2011. All analyzed viruses evolved from the lineage of subtype H5N1 viruses introduced into Egypt in 2006; we found minimal evidence of reassortment and no exotic introductions. The hemagglutinin genes of the viruses from 2011 formed a monophyletic group within clade 2.2.1 that also included human viruses from 2009 and 2010 and contemporary viruses from poultry; this finding is consistent with zoonotic transmission. Although molecular markers suggestive of decreased susceptibility to antiviral drugs were detected sporadically in the neuraminidase and matrix 2 proteins, functional neuraminidase inhibition assays did not identify resistant viruses. No other mutations suggesting a change in the threat to public health were detected in the viral proteomes. However, a comparison of representative subtype H5N1 viruses from 2011 with older subtype H5N1 viruses from Egypt revealed substantial antigenic drift.

A molecular investigative approach to an outbreak of acute hemorrhagic conjunctivitis in Egypt, October 2010.

BACKGROUND: During October 2010, Egypt reported an outbreak of acute hemorrhagic conjunctivitis (AHC). A total of 1831 cases were reported from three governorates; 1703 cases in El Daqahliya, 92 cases in Port Said, and 36 in Damietta. The purpose of this study was to identify and characterize the causative agent associated with this outbreak. METHODS: The U.S. Naval Medical Research Unit No.3 (NAMRU-3) was contacted by the Egyptian Ministry of Health and Population to perform diagnostic laboratory testing on eighteen conjunctival swabs from patients with conjunctivitis from El Daqahliya Governorate. Conjunctival swabs were tested by molecular methods for human adenovirus (HAdV) and enteroviruses (EV). Virus isolation was performed; the isolated virus was further characterized by molecular typing and phylogenetic analysis. RESULTS: The majority of the samples (17/18) were positive for enterovirus and all were negative for HAdV. Molecular typing and sequencing of the isolated virus revealed the presence of coxsackievirus A24 variant. Phylogenetic analysis based on the VP1 and 3C regions demonstrated that the Egyptian viruses belonged to Genotype IV and are closely related to coxsackievirus A24 variant, reported in a similar outbreak in China in August 2010. CONCLUSIONS: This study strongly suggests that coxsackievirus A24 variant was associated with the acute hemorrhagic conjunctivitis outbreak reported in Egypt in October 2010. There is a possibility that the same strain of CV-A24v was implicated in the AHC outbreaks in both China and Egypt in 2010.

Viral etiologies of lower respiratory tract infections among Egyptian children under five years of age.

BACKGROUND: Lower respiratory tract infections (LRTI) are responsible for a considerable number of deaths among children, particularly in developing countries. In Egypt and the Middle East region, there is a lack of data regarding the viral causes of LRTI. In this study, we aimed to identify the relative prevalence of various respiratory viruses that contribute to LRTIs in young children. Although, nucleic acid-based methods have gained importance as a sensitive tool to determine the viral infections, their use is limited because of their prohibitive cost in low-income countries. Therefore, we applied three different laboratory methods, and presented the different virus prevalence patterns detected by each method. METHODS: We collected nasopharyngeal aspirate samples, demographic data and, clinical data from 450 children under five years of age who presented with LRTI at Abou El Reesh hospital in Cairo during a one-year period. To identify the viral causes of the LRTI we used direct fluorescence assay, real-time reverse-transcriptase polymerase chain reaction (rt-RT-PCR), and shell vial culture. We tested for eight major respiratory viruses. RESULTS: Two hundred sixty-nine patients (59.9%) had a viral infection, among which 10.8% had a co-infection with two or more viruses. By all three methods, respiratory syncytial virus (RSV) was the most predominant, and parainfluenza virus type 2 (HPIV-2), influenza B virus (FLUBV) were the least predominant. Other viral prevalence patterns differed according to the detection method used. The distribution of various viruses among different age groups and seasonal distribution of the viruses were also determined. CONCLUSIONS: RSV and human adenovirus were the most common respiratory viruses detected by rt-RT-PCR. Co-infections were found to be frequent among children and the vast majority of co-infections were detected by nucleic acid-based detection assays.

Effects of hand hygiene campaigns on incidence of laboratory-confirmed influenza and absenteeism in schoolchildren, Cairo, Egypt.

To evaluate the effectiveness of an intensive hand hygiene campaign on reducing absenteeism caused by influenza-like illness (ILI), diarrhea, conjunctivitis, and laboratory-confirmed influenza, we conducted a randomized control trial in 60 elementary schools in Cairo, Egypt. Children in the intervention schools were required to wash hands twice each day, and health messages were provided through entertainment activities. Data were collected on student absenteeism and reasons for illness. School nurses collected nasal swabs from students with ILI, which were tested by using a qualitative diagnostic test for influenza A and B. Compared with results for the control group, in the intervention group, overall absences caused by ILI, diarrhea, conjunctivitis, and laboratory-confirmed influenza were reduced by 40%, 30%, 67%, and 50%, respectively (p<0.0001 for each illness). An intensive hand hygiene campaign was effective in reducing absenteeism caused by these illnesses.

Crimean-Congo hemorrhagic fever, Afghanistan, 2009.

In response to an outbreak of Crimean-Congo hemorrhagic fever in western Afghanistan, we measured immunoglobulin G seroprevalence among household members and their animals. Seroprevalence was 11.2% and 75.0% in humans (n = 330) and livestock (n = 132), respectively. Persons with frequent exposure to cattle had an elevated risk of being immunoglobulin G positive.

Correction: Rodent-borne infections in rural Ghanaian farming communities.

[This corrects the article DOI: 10.1371/journal.pone.0215224.].

Rodent-borne infections in rural Ghanaian farming communities.

Rodents serve as reservoirs and/or vectors for several human infections of high morbidity and mortality in the tropics. Population growth and demographic shifts over the years have increased contact with these mammals, thereby increasing opportunities for disease transmission. In Africa, the burden of rodent-borne diseases is not well described. To investigate human seroprevalence of selected rodent-borne pathogens, sera from 657 healthy adults in ten rural communities in Ghana were analyzed. An in-house enzyme-linked immunosorbent assay (ELISA), for immunoglobulin G (IgG) antibodies to Lassa virus was positive in 34 (5%) of the human samples. Using commercial kits, antibodies to hantavirus serotypes, Puumala and Dobrava, and Leptospira bacteria were detected in 11%, 12% and 21% of the human samples, respectively. Forty percent of residents in rural farming communities in Ghana have measurable antibodies to at least one of the rodent-borne pathogens tested, including antibodies to viral hemorrhagic fever viruses. The high seroprevalence found in rural Ghana to rodent-borne pathogens associated with both sporadic cases and larger disease outbreaks will help define disease threats and inform public health policy to reduce disease burden in underserved populations and deter larger outbreaks.

Outbreak of sandfly fever in central Iraq, September 2007.

An outbreak of nonspecific febrile illnesses occurred among U.S. Army troops in September 2007 at a remote, newly established, rural-situated patrol base, south of Baghdad, Iraq. Soldiers displayed an acute flu-like syndrome with symptoms of fever, headache, malaise, and myalgia. A total of 14 cases was identified and treated presumptively as query fever. Subsequent convalescent serum specimens confirmed 13 (92.9%) positive for sandfly Sicilian virus and 3 (21.4%) positive for Coxiella burnetii, with two positive for both. One sandfly Sicilian virus case tested positive for Brucella spp. This outbreak emphasizes the potential for multiple simultaneous disease exposures to endemic diseases in nonindigenous military personnel at remote military locations in Iraq. Recommendations include increased theater disease surveillance, medical training, and vector control.

A cross-sectional household cluster serosurvey of hepatitis C virus antibodies in an urban slum of Cairo, Egypt in 2004.

BACKGROUND: Hepatitis C Virus (HCV) continues to be a burden to the Egyptian population and its economy. Despite all efforts, the prevalence of infection continues to be one of the highest in the world. The high national prevalence has been attributed to unintentional, nosocomial spread during an anti-schistosomiasis campaign conducted in the 1970's, but that does not fully explain the persistent infection rates. Work is needed to further clarify risk associations. A serosurvey was performed in Manshiet Nasser, a slum in Cairo sometimes referred to as Mokattem Hills where a primary occupation is garbage collection and sorting, to characterize potential risk factors for infection. METHODS: Following a detailed mapping and census of the area, a cluster sampling was performed and demographic and risk behavior data and a blood sample were collected from subjects older than 6 months. Blood samples were tested using 4th generation anti-HCV EIA kit. RESULTS: The slum was estimated to house 45,000 residents. Blood samples were obtained from 2169 subjects. The age adjusted anti-HCV seroprevalence was 9.1 %. Participants with HCV antibodies were more likely to be male, heads of households, subjects without formal education, and those with a lower standard of living. After adjustment of all prevalence ratios (aPR) for age, the following risk factors were significantly associated with higher risk of HCV infection: Garbage collection (aPR 1.5), history of blood transfusion (aPR 2.0), tattooing (aPR 1.4), history of schistosomiasis (aPR 1.5), and use of traditional latrines (aPR 2.0) or pits for sanitation (aPR 1.3). The results of the multivariate analysis indicated that age (p < 0.01), history of schistosomiasis (p < 0.05), garbage sorting (p = 0.05), blood transfusions (<0.001), and the use of traditional latrines for sanitation (p < 0.01) were significantly associated with infection. CONCLUSION: While HCV prevalence among those over 30 could be attributed to anti-schistosomiasis injections, the prevalence in younger age indicates ongoing transmission. Although specific behavioral risks were not identified, HCV infection appears to be an occupational hazard of garbage collection and sorting in this environment. Given the large reservoir of HCV infection in the population, further effort needs to be made to identify and mitigate new infections.

Chikungunya outbreak in Al-Hudaydah, Yemen, 2011: epidemiological characterization and key lessons learned for early detection and control.

Little is known about the occurrence of chikungunya fever in the Eastern Mediterranean Region of the World Health Organization (WHO). In January 2011, the Ministry of Public Health and Population (MoPH&P) of Yemen reported to WHO an increasing number of "dengue-like" acute febrile illnesses of unknown origin from one of its coastal governorates. An epidemiological investigation was conducted in Al-Hudaydah governorate between 23 and 26 January 2011 by a joint team of WHO, the MoPH&P of Yemen and the U.S. Naval Medical Research Unit (NAMRU-3) in Cairo, Egypt. The investigation led to the detection of an outbreak of chikungunya in Yemen which was the first time ever from any of the 22 countries in the Eastern Mediterranean Region of WHO. Appropriate public health control measures were strengthened following the investigation, and the outbreak was contained. This paper provides a short description of the outbreak and its epidemiological characteristics and highlights the important lessons that were learned for early detection and control of chikungunya in countries where competent vectors for transmission of the virus exist.

Serosurvey for zoonotic viral and bacterial pathogens among slaughtered livestock in Egypt.

INTRODUCTION: Zoonotic diseases are an important cause of human morbidity and mortality. Animal populations at locations with high risk of transmission of zoonotic pathogens offer an opportunity to study viral and bacterial pathogens of veterinary and public health concern. METHODS: Blood samples were collected from domestic and imported livestock slaughtered at the Muneeb abattoir in central Egypt in 2009. Samples were collected from cattle (n=161), buffalo (n=153), sheep (n=174), and camels (n=10). Samples were tested for antibodies against Leptospira spp. by a microscopy agglutination test, Coxiella burnetii by enzyme immunoassay, Brucella spp. by standard tube agglutination, and Rift Valley Fever virus (RVFV), Crimean-Congo hemorrhagic fever virus (CCHFV), sandfly fever Sicilian virus (SFSV), and sandfly fever Naples virus (SFNV) by enzyme-linked immunosorbent assay. RESULTS: Antibodies against Leptospira spp. were identified in 64 (40%) cattle, 45 (29%) buffalo, 71 (41%) sheep, and five (50%) camels; antibodies against C. burnetii in six (4%) buffalo, 14 (8%) sheep, and seven (70%) camels; and antibodies against Brucella spp. in 12 (8%) cattle, one (1%) buffalo, seven (4%) sheep, and one (10%) camel. Antibodies against RVFV were detected in two (1%) cattle and five (3%) buffalo, and antibodies against CCHFV in one (1%) cow. No antibodies against SFSV or SFNV were detected in any species. DISCUSSION: RESULTS indicate that livestock have been exposed to a number of pathogens, although care must be taken with interpretation. It is not possible to determine whether antibodies against Leptospira spp. and RVFV in cattle and buffalo are due to prior vaccination or natural exposure. Similarly, antibodies identified in animals less than 6 months of age may be maternal antibodies transferred through colostrum rather than evidence of prior exposure. RESULTS provide baseline evidence to indicate that surveillance within animal populations may be a useful tool to monitor the circulation of pathogens of veterinary and public health concern in Egypt.

Hemorrhagic fever with renal syndrome and Crimean-Congo hemorrhagic fever as causes of acute undifferentiated febrile illness in Bulgaria.

Hemorrhagic fever with renal syndrome (HFRS) and Crimean-Congo hemorrhagic fever (CCHF) are the 2 widespread viral hemorrhagic fevers occurring in Europe. HFRS is distributed throughout Europe, and CCHF has been reported mainly on the Balkan Peninsula and Russia. Both hemorrhagic fevers are endemic in Bulgaria. We investigated to what extent acute undifferentiated febrile illness in Bulgaria could be due to hantaviruses or to CCHF virus. Using enzyme-linked immunosorbent assays (ELISAs), we tested serum samples from 527 patients with acute febrile illness for antibodies against hantaviruses and CCHF virus. Immunoglobulin M (IgM) antibodies against hantaviruses were detected in 15 (2.8%) of the patients. Of the 15 hantavirus-positive patients, 8 (1.5%) were positive for Dobrava virus (DOBV), 5 (0.9%) were positive for Puumala virus (PUUV), and the remaining 2 were positive for both hantaviruses. A plaque reduction neutralization test (PRNT) confirmed 4 of the 10 DOBV-positive samples. PRNT was negative for all PUUV-positive samples. Serologic evidence of recent CCHF virus infection was found in 13 (2.5%) of the patients. Interestingly, HFRS and CCHF were not only detected in well-known endemic areas of Bulgaria but also in nonendemic regions. Our results suggested that in endemic countries, CCHF and/or HFRS might appear as a nonspecific febrile illness in a certain proportion of patients. Physicians must be aware of possible viral hemorrhagic fever cases, even if hemorrhages or renal impairment are not manifested.

Seroepidemiologic survey for Coxiella burnetii among US military personnel deployed to Southwest and Central Asia in 2005.

We used a seroepidemiologic study to estimate Q fever (Coxiella burnetii) seroprevalence, seroincidence, and risk factors for seroconversion in two deployed military populations in 2005. The first study group resided in an area with a known Q fever outbreak history (Al Asad, Iraq). Of this population, 7.2% seroconverted for an incidence rate of 10.6 seroconversions per 1,000 person-months. The second population included personnel transiting through Qatar on mid-deployment leave from southwest/central Asia. In this group, we found 2.1% prevalence with 0.92 seroconversions per 1,000 person-months. However, no significant risk factors for Q fever seroconversion were found in either population.

A high diversity of Eurasian lineage low pathogenicity avian influenza A viruses circulate among wild birds sampled in Egypt.

Surveillance for influenza A viruses in wild birds has increased substantially as part of efforts to control the global movement of highly pathogenic avian influenza A (H5N1) virus. Studies conducted in Egypt from 2003 to 2007 to monitor birds for H5N1 identified multiple subtypes of low pathogenicity avian influenza A viruses isolated primarily from migratory waterfowl collected in the Nile Delta. Phylogenetic analysis of 28 viral genomes was performed to estimate their nearest ancestors and identify possible reassortants. Migratory flyway patterns were included in the analysis to assess gene flow between overlapping flyways. Overall, the viruses were most closely related to Eurasian, African and/or Central Asian lineage low pathogenicity viruses and belonged to 15 different subtypes. A subset of the internal genes seemed to originate from specific flyways (Black Sea-Mediterranean, East African-West Asian). The remaining genes were derived from a mixture of viruses broadly distributed across as many as 4 different flyways suggesting the importance of the Nile Delta for virus dispersal. Molecular clock date estimates suggested that the time to the nearest common ancestor of all viruses analyzed ranged from 5 to 10 years, indicating frequent genetic exchange with viruses sampled elsewhere. The intersection of multiple migratory bird flyways and the resulting diversity of influenza virus gene lineages in the Nile Delta create conditions favoring reassortment, as evident from the gene constellations identified by this study. In conclusion, we present for the first time a comprehensive phylogenetic analysis of full genome sequences from low pathogenic avian influenza viruses circulating in Egypt, underscoring the significance of the region for viral reassortment and the potential emergence of novel avian influenza A viruses, as well as representing a highly diverse influenza A virus gene pool that merits continued monitoring.

Comparison of direct fluorescence assay and real-time rt-PCR as diagnostics for respiratory syncytial virus in young children.

Respiratory syncytial virus (RSV) is the major cause of lower respiratory tract infections in children worldwide. Early detection of RSV is critical to initiate proper care. Two methods, the direct fluorescence assay (DFA) and the real-time reverse-transcription polymerase chain reaction (rt-RT-PCR), that are used for RSV detection were compared. A total of 451 nasopharyngeal aspirates from children 5 years of age or less were tested for RSV using both methods. The overall prevalence rate of the RSV among the children was 23.7% with a significantly higher prevalence among children under the age of 6 months of age when compared to other age groups. The sensitivity of DFA in comparison to rt-RT-PCR was highest (86%) during the first 3 days of symptoms onset and decreased gradually till it reached 65% after the first week. The specificity of DFA in comparison to rt-RT-PCR ranged between 99 and 100% irrespective of the date of collection. We concluded that, although the rt-RT-PCR is more sensitive for RSV detection, the DFA offers a reliable point-of-care alternative detection method especially during the first few days of illness.

Dengue hemorrhagic fever outbreak in children in Port Sudan.

Dengue fever (DF)/dengue hemorrhagic fever (DHF) has emerged as a global public health problem with countries in Asia and the Pacific sharing more than 70% of the disease burden. In 2004-2005 a total of 312 cases admitted to Pediatric and Sea Port Hospitals in Port Sudan were clinically diagnosed as DHF. The mortality rate recorded was 3.8% (n=12). Of the cases 73.4% were patients 5-15 years of age. A total of 91.2% of cases were admitted during May and June 2005 with 49.4% residing in the eastern region of Port Sudan. Dengue shock syndrome was observed in 37 of 312 (11.9%). All patients had thrombocytopenia with platelets count ranged from <100,000 to <150,000 cell/mm³. Of the 40 sera tested using RAPID-cassette test in the Khartoum Central Public Health Lab, 36 (90%) were dengue IgM positive. A subset of these sera (n=23) were sent to NAMRU-3 and confirmed by IgM-capture ELISA; 9 of 23 were PCR positive for dengue serotype 3.