http://www.keelpno.gr [email protected] VOL. 25 E_bulletin Hellenic Center for Disease Control and Prevention Agrafon 3- 5, Maroussi, 15123, Tel: +30 210 5212000, [email protected], http://www.keelpno.gr March 2013 ISSN 1792-9016 Vol. 25/ Year 3rd MINISTRY OF HEALTH HCDCP HELLENIC CENTER FOR DISEASE CONTROL & PREVENTION MINISTRY OF HEALTH This issue includes an interview with Mr A. Gikas, Professor of Pathology-Infectious Diseases at the University of Crete. The professor refers to the great contribution of infectious diseases to the field of medicine and highlights the critical involvement of doctors in relation to epidemiological surveillance and the mandatory notification system for effective monitoring of diseases. More on page 37 Two years of the e-bulletin! HCDCP’s e-bulletin has recently completed 2 years of web trafficking! More on page 43 Q FEVER Q fever is a zoonosis caused by the Coxiella burnetii, a Gram-negative obligate intracellular pathogen responsible for acute and chronic clinical manifestations of the disease. Coxiella burnetii was discovered in 1937 and classified as a new species of the family Rickettsiaceae. Recent phylogenetic studies, mainly based on 16S rRNA analysis, have shown that C.burnetii belongs to the Coxiel- laceae family, within the order of Legionellales and the gamma subdivision of Proteobacteria. Q fever is considered to be a recently emerging disease. It is believed that the increasing incidence in diagnosis of Q fever cases is the result of a combination of actual increasing numbers of Q fever cases (re-appear- ance), a growing interest among physicians, better laboratory diagnosis and the availability of clinical and epidemiological data. Nevertheless, the true incidence of Q fever (a relatively rare disease, which is a mandatory modifiable disease in only a few countries in the world)can- not be estimated. Usually a person will develop a primary infection with a mild symptomatol- ogy (’flu-like syndrome) or pneumonia or hepatitis, etc., after exposure to C. burnetiiby inhalation and incubation of 10–17 days. One of the main characteristics of Q fever is the evolution (2–5% of patients) to achronic form of the disease, mainly endocarditis and vascular infection. Diagnosis of the disease is based on serology, cultivation and detec- tion of DNA in blood or tissue, using polymerase chain reaction (PCR). The therapeutic treatment consists of doxycycline with hydroxychloroquine and sometimes surgery in the case of chronic disease, and doxycycline in the case of acute disease. Q fever was almost unknown to Greek physicians until the mid-1990s. Nowadays, because of the experience gained inboth Greece and interna- tionally, the vast majority of Greek clinicians include Q fever in the differ- ential diagnosis of atypical pneumonia, prolonged febrile, culture-negative endocarditis, chronic fatigue, etc. In our country the disease is included among the mandatory notifiable diseases. Under-notification, subclinical forms of the disease, a typical clinical pre- sentation and often a lack of interest are factors contributing to very low incidence rates. This issue of the Hellenic Center for Disease Control and Prevention (HCD- CP)e-bulletin, with the participation of distinguished and experienced sci- entists in the field, is aimed at facilitating a better understanding of the disease, elucidating of the ‘gray zones ’in laboratory diagnosis, updating information on the therapeutic evolution of the disease and finally provid- ing an estimation of the magnitude of the problem in our country, compar- ing its incidence with relevant international reports. Professor Achilleas Gikas Contents Main article: Descriptive epidemiology of Q fever in Greece, 2004-2012 2 Surveillance data 7 Public health news 11 Invited articles 13 HCDCP’ s departments activities 33 Interesting activities 34 Future conferences 36 Interview 37 Myths and truths 39 Outbreaks around the world 42 Quiz of the month 44 Highlights
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VOL. 25
E_bulletinHellenic Center for Disease Control and PreventionAgrafon 3- 5, Maroussi, 15123, Tel: +30 210 5212000,[email protected], http://www.keelpno.gr
March 2013 ISSN 1792-9016Vol. 25/ Year 3rd
MINISTRY OF HEALTH
HCDCP
HELLENIC CENTER FORDISEASE CONTROL & PREVENTION
MINISTRY OF HEALTH
This issue includes an interview with Mr A. Gikas, Professor of Pathology-Infectious Diseases at the University of Crete. The professor refers to the great contribution of infectious diseases to the field of medicine and highlights the critical involvement of doctors in relation to epidemiological surveillance and the mandatory notification system for effective monitoring of diseases.
More on page 37
Two years of the e-bulletin! HCDCP’s e-bulletin has recently completed 2 years of web trafficking!More on page 43
Q FEVER
Q fever is a zoonosis caused by the Coxiella burnetii, a Gram-negative obligate intracellular pathogen responsible for acute and chronic clinical manifestations of the disease.Coxiella burnetii was discovered in 1937 and classified as a new species of the family Rickettsiaceae. Recent phylogenetic studies, mainly based on 16S rRNA analysis, have shown that C.burnetii belongs to the Coxiel-laceae family, within the order of Legionellales and the gamma subdivision of Proteobacteria.Q fever is considered to be a recently emerging disease. It is believed that the increasing incidence in diagnosis of Q fever cases is the result of a combination of actual increasing numbers of Q fever cases (re-appear-ance), a growing interest among physicians, better laboratory diagnosis and the availability of clinical and epidemiological data.Nevertheless, the true incidence of Q fever (a relatively rare disease, which is a mandatory modifiable disease in only a few countries in the world)can-not be estimated.Usually a person will develop a primary infection with a mild symptomatol-ogy (’flu-like syndrome) or pneumonia or hepatitis, etc., after exposure to C. burnetiiby inhalation and incubation of 10–17 days.One of the main characteristics of Q fever is the evolution (2–5% of patients) to achronic form of the disease, mainly endocarditis and vascular infection.Diagnosis of the disease is based on serology, cultivation and detec-tion of DNA in blood or tissue, using polymerase chain reaction (PCR). The therapeutic treatment consists of doxycycline with hydroxychloroquine and sometimes surgery in the case of chronic disease, and doxycycline in the case of acute disease.Q fever was almost unknown to Greek physicians until the mid-1990s.Nowadays, because of the experience gained inboth Greece and interna-tionally, the vast majority of Greek clinicians include Q fever in the differ-ential diagnosis of atypical pneumonia, prolonged febrile, culture-negative endocarditis, chronic fatigue, etc.In our country the disease is included among the mandatory notifiable diseases.Under-notification, subclinical forms of the disease, a typical clinical pre-sentation and often a lack of interest are factors contributing to very low incidence rates.This issue of the Hellenic Center for Disease Control and Prevention (HCD-CP)e-bulletin, with the participation of distinguished and experienced sci-entists in the field, is aimed at facilitating a better understanding of the disease, elucidating of the ‘gray zones ’in laboratory diagnosis, updating information on the therapeutic evolution of the disease and finally provid-ing an estimation of the magnitude of the problem in our country, compar-ing its incidence with relevant international reports.
Professor Achilleas Gikas
Contents
Main article: Descriptive epidemiology of Q fever in Greece, 2004-2012 2
Surveillance data 7
Public health news 11
Invited articles 13
HCDCP’ s departments activities 33
Interesting activities 34
Future conferences 36
Interview 37
Myths and truths 39
Outbreaks around the world 42
Quiz of the month 44
Highlights
Main article 2
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Descriptive epidemiology of Q fever in Greece, 2004-2012
PrefaceQ fever is an infectious microbial disease; the causative agent is Coxiella burnetii. The acute form manifests with generalized ’flu-like symptoms and varies from subclinical infection to serious disease. The chronic form is commonly associated with endocarditis, mainly in individuals with underlying cardiovalvulopathy. The causative agent is found in animals such as sheep, goats, cattle, felids and rodents. Placentas, genital secretions and other parturition products are heavily contaminated and contribute to the dissemination of the micro-organism, which is highly resistant in the environment. Ticks can also harbor C. burnetii. Human infection is usually airborne through inhalation of infected aerosols. Transmission also occurs by consumption of unpasteurized or non-adequately heated milk products, by blood transfusion and organ transplantation and by tick bite.
Disease surveillanceQ fever is a mandatory notifiable disease. Practitioners must report cases to the Hellenic Center for Disease Control and Prevention (HCDCP). According to the European Center for Disease Control, the definition of a case needs to fulfill both clinical and laboratory criteria.
Clinical criteria At least one of the following: fever, pneumonia, hepatitis.
Laboratory findingsIsolation of the pathogen, detection of the nucleic acid of the pathogen in a clinical specimen or a positive reaction for specific antibodies against C. burnetii (IgG or IgM phase II). The Laboratory of Clinical Bacteriology, Parasitology, Zoonoses and Geographical Medicine, of the Medical School at Crete University, plays a very important role in the laboratory investigation of Q fever cases in Greece.During interpretation of epidemiological data, it has to be borne in mind that an unknown percentage of cases goes unreported, as happens with all notifiable diseases. Notably in some countries, in Europe and elsewhere, Q fever is not under mandatory surveillance.
Temporal trendThe mean annual incidence during 2004-2012 was 0.033 cases/100,000 head of population (range 0.009-0.102/100,000) (Figure 1).Α distinct peak was apparent in 2012, and in the same year 63.6% (7/11) of cases were residents of the Larisa prefecture in the region of Thessaly. Temporal or spatial variation of the incidence of the disease can be attributed to differences in the further laboratory examination of suspicious cases including Q fever in the investigation panel. Notably, there are no indications from animal surveillance of an increase in animal cases at a national or regional level in 2004-2012. Despite the annual relative variation in incidence, the national incidence remains at low levels in absolute terms. In the European Union (EU) in 2009, 0.600 cases/100,000 were recorded. Holland presented the highest incidence, with 9.800 cases/100,000, where over the last few years there has been a continuous long-term outbreak in animals.
Age/sex distributionThe mean age of cases has been 48.2 years (median 51 years, range 16-88 years) (Figure 2). In Greece, the most affected age group has been between 45 and 64 years (37% of cases), as is also the case in the EU. The majority of cases have been male (male/female ratio=1.5), again in accordance with the EU (male/female=1.58).
SeasonalityThe highest monthly incidence is recorded in June (mean monthly incidence 0.050 cases/100,000) (Figure 3). In the EU a similar seasonality appears (42% of cases in June and July). The end of spring–mid-summer bears the highest incidence and is related to the period of parturition in sheep and goats (April-May) and an increased load of the pathogen in the environment. Climatic conditions, i.e. especially strong winds, may play a significant role in the long-distance dissemination of the pathogen to large parts of the population.
Geographical occurrenceThessaly presents the highest incidence for 2004-2012, with a mean annual value of 0.152 cases/100,000; the Peloponnese comes second with 0.096 cases/100,000 (Figure 4). Regional variations must be interpreted with caution because they can be partly explained by a different sensitivity of practitioners to proceeding with further laboratory research that includes Q fever. For instance, most of the cases in Thessaly-Larisa were sent to the Medical School University Hospital of Larisa for diagnosis. Risk factorsA high-risk profession was recorded in 33% of cases (9/28) (Figure 5) among these 33.3% (3/9) were farmers, 55.6% (5/9) livestock breeders and 11.1% (1/9) other high-risk occupations, such as students at a veterinary-forestry school.
DiscussionQ fever is a disease that can easily be misdiagnosed because of its frequently subclinical character. However, smaller and larger outbreaks are possible as large parts of the population may be effectively exposed via the airborne transmission route and the pathogen remains remarkably stable in the external environment. High-risk groups for infection are those who frequently come in close contact with animals, mainly sheep and goats. The most vulnerable population for the more serious forms of disease are those with underlying cardiovalvular disease, pregnant women and immunodeficient individuals. Prompt diagnosis and initiation of treatment reduces the possibility of chronic Q fever with the complication of endocarditis. Abortions, premature births and birth of underweight newborns may manifest in pregnant women. Often the incidence in humans is an indicator of the rate of infection in animals, where the signs and symptoms often go unnoticed. On the other hand, the mild and self-limiting nature of the disease in humans and its similarity with influenza impedes further investigation and correct diagnosis. Often new cases are found when Q fever is included in the laboratory investigation panel in patients with a history of contact with animals and compatible clinical presentation. Preventative measures in animals, which include appropriate management of parturition and abortion products (prevention of exposure to strong winds, destruction with fire and disinfection) and therapeutic or preventive antibiotic administration, are often implemented to maximize meat and milk production and prevent the spread of the disease in humans.
Main article 4
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Figure 1:
Q fever incidence, Greece 2004-2012
0,000
0,050
0,100
0,150
0,200
0,250
0,300
0,350
0,400
0,450
0,500
2004 2005 2006 2007 2008 2009 2010 2011 2012
Year
Case
s /
100,0
00
Figure 2:
Q fever cases by sex and age group, Greece 2004-2012
0
1
2
3
4
5
6
7
8
0-4 5-14 15-24 25-44 45-64 65-max
Age group
Case
s
Male
Female
Figure 3:
Q fever seasonal distribution, Greece 2004-2012
0,000
0,001
0,002
0,003
0,004
0,005
0,006
Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec
Onset of disease
Case
s /
100,0
00
Figure 4. Annual incidence of Q fever by region, Greece 2004-2012
Surveillance data
6
http://www.keelpno.gr
Figure 5:
High risk occupation, Q fever, Greece 2004-2012
67%
11%
18%
4%
Not a high risk occupation
Farmer
Livestock breeder
Other
Georgios Dougas, DVM, Office of Zoonoses, HCDCP
Table 1: Number of notified cases in February 2013, median number and range of notified cases in February for the years 2004−2012, Mandatory Notification
System, Greece
Disease Number of notified cases
February 2013
Median numberFebruary
2004−2012
Min. number February
2004-2012
Max. number February
2004-2012
Botulism 0 0 0 1Chickenpox with complications 2 2 0 4Anthrax 0 0 0 1Brucellosis 9 8 2 24Diphtheria 0 0 0 0Arbo-viral infections 0 0 0 0Malaria 1 0 0 3Rubella 0 0 0 3Smallpox 0 0 0 0Echinococcosis 0 1 0 3Hepatitis Α 11 9 3 27Hepatitis B, acute & HBsAg(+) in infants <12 months 1 6 1 13
Hepatitis C, acute & confirmed anti-HCV positive (1st diagnosis) 1 1 0 7
Measles 1 0 0 75Haemorrhagic fever 0 0 0 0Pertussis 5 1 0 26Legionellosis 1 0 0 3Leishmaniasis 2 3 1 7Leptospirosis 1 0 0 3Listeriosis 1 0 0 1EHEC infection 0 0 0 1Rabies 0 0 0 0Melioidosis/glanders 0 0 0 0Meningitis
aseptic 14 9 6 23bacterial (except meningococcal disease) 12 17 11 21
unknown etiology 0 1 0 4Meningococcal disease 8 11 5 22Plague 0 0 0 0Mumps 0 0 0 4
Poliomyelitis 0 0 0 0
Q fever 1 0 0 1Salmonellosis (non-typhoid/paratyphoid) 11 20 10 42
Shigellosis 7 1 0 3
Severe acute respiratory syndrome 0 0 0 0
Congenital rubella 0 0 0 0
Congenital syphilis 0 0 0 0
Congenital toxoplasmosis 0 0 0 0Cluster of foodborne/waterborne disease cases 4 1 0 9
Τetanus/neonatal tetanus 0 0 0 2
Tularaemia 0 0 0 0
Trichinosis 0 0 0 3
Typhoid fever/paratyphoid 0 1 0 3
Tuberculosis 34 48 31 61
Cholera 0 0 0 0
Surveillance data
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Table 2:Number of notified cases by place of residence (region), Mandatory Notification System, Greece, February 2013 (place of residence is defined
according to the home address of the patient).
Disease Number of notified cases
Region
Eas
tern
Mac
edonia
and T
hra
ce
Cen
tral
Mac
edonia
Wes
tern
Mac
edonia
Epirus
Thes
salia
Ionia
n isl
ands
Wes
tern
Gre
ece
Ste
rea
Gre
ece
Att
ica
Pelo
ponnes
e
Nort
her
n A
egea
n
South
ern A
egea
n
Cre
te
Unkn
ow
n
Chickenpox with complications 0 0 0 0 1 0 0 0 0 0 0 0 0 1
Brucellosis 1 0 0 1 2 0 2 3 0 0 0 0 0 0
Malaria 0 0 0 0 0 0 0 1 0 0 0 0 0 0
Hepatitis Α 1 0 0 0 1 1 0 1 4 3 0 0 0 0
Hepatitis B, acute & HBsAg(+) in infants <12 months 0 0 0 0 0 0 0 1 0 0 0 0 0 0
Hepatitis C, acute & confirmed anti=HCV positive (1st diagnosis) 0 0 0 0 1 0 0 0 0 0 0 0 0 0
Measles 0 0 0 0 0 0 0 0 1 0 0 0 0 0
Pertussis 0 0 0 0 0 0 0 1 3 1 0 0 0 0
Legionellosis 0 0 0 0 0 0 0 0 1 0 0 0 0 0
Leishmaniasis 0 1 0 0 0 0 0 0 1 0 0 0 0 0
Leptospirosis 0 0 0 0 0 0 0 1 0 0 0 0 0 0
Listeriosis 0 0 0 0 0 0 0 0 1 0 0 0 0 0
Meningitis
aseptic 3 0 0 0 3 1 3 0 3 1 0 0 0 0
bacterial (except meningococcal disease) 0 1 0 0 0 0 1 1 6 2 0 0 1 0
Meningococcal disease 1 2 0 0 2 0 0 0 2 0 0 1 0 0
Q fever 0 0 0 0 1 0 0 0 0 0 0 0 0 0
Salmonellosis (non-typhoid/paratyphoid) 1 2 0 0 0 0 0 3 4 1 0 0 0 0
Shigellosis 0 0 0 0 0 4 0 1 2 0 0 0 0 0
Cluster of foodborne/waterborne disease cases 0 2 0 0 0 1 0 0 1 0 0 0 0 0
Tuberculosis 1 4 0 4 1 1 2 3 9 5 0 0 2 2
Table 3: Number of notified cases by age group and gender, Mandatory Notification System, Greece, February 2013 (M: male; F: female)
Disease Number of notified cases by age group (years) and gender
<1 1-4 5-14 15-24 25-34 35-44 45-54 55-64 65+ Un.
M F M F M F M F M F M F M F M F M F M F
Chickenpox with complications 0 0 0 0 0 0 0 0 0 0 2 0 0 0 0 0 0 0 0 0
Brucellosis 0 0 0 0 0 0 1 0 1 0 1 1 3 0 0 1 1 0 0 0
Malaria 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0
Hepatitis Α 0 0 0 0 2 2 1 1 0 1 1 2 0 0 0 1 0 0 0 0
Hepatitis B, acute & HBsAg(+) in infants <12 months
0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
Hepatitis C, acute & confirmed anti-HCV positive (1st diagnosis)
0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0
Measles 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0
Pertussis 4 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
Legionellosis 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0
Leishmaniasis 0 0 0 0 0 1 0 0 1 0 0 0 0 0 0 0 0 0 0 0
Leptospirosis 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0
Listeriosis 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0
Meningitis
aseptic 3 0 0 2 2 1 1 0 0 0 2 0 0 0 0 0 1 1 1 0
bacterial (except meningococcal disease) 0 1 1 1 1 0 0 0 0 1 0 0 0 1 0 3 1 2 0 0
Meningococcal disease 1 1 1 3 0 0 0 1 1 0 0 0 0 0 0 0 0 0 0 0
Q fever 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0
Salmonellosis (non-typhoid/paratyphoid) 0 0 0 2 3 3 0 1 0 0 0 0 1 0 1 0 0 0 0 0
Shigellosis 1 0 0 3 1 2 0 0 0 0 0 0 0 0 0 0 0 0 0 0
Tuberculosis 0 0 0 0 2 2 5 1 1 1 2 0 4 0 2 0 9 5 0 0
Public health news
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The datapresentedare derived from the Mandatory Notification System (MNS) of the Hellenic Centerfor Disease Control and Prevention (HCDCP). Forty-five infectious diseases are included in the list of the mandatory notifiable diseases in Greece. Notification forms and case definitions can be found onthe HCDCP website (www.keelpno.gr).It should be noted that the data for February2013 are provisional, and maybe slightly modified/corrected in the future, and also that data interpretation should be made with caution as there are indications of under-reporting inthe system.
Department of Epidemiological Surveillance and Intervention
EUROPEAN IMMUNIZATION WEEK 22-27 April 2013
‘Get vaccinated /Protect your world’
The European Immunization Week (EIW) is an initiative led and co-ordinated by the World Health Organization (WHO) Europe and implemented by member states of the European region. For one week in April, countries across the region unite under the EIW slogan, ‘Prevent. Protect. Immunize’ and carry out activities to inform audiences. EIW will take place on 22-27 April 2013 and aims to promote one of the world’s most powerful tools for public health, vaccines, which protect and ‘immunize’ people of all ages. The ultimate goal of EIW is that more and more people will be protected from vaccine-preventable diseases. Immunization is one of the most successful and cost-effective health interventions and prevents between 2 and 3 million deaths every year. From infants to senior citizens, immunization protects against diseases such as diphtheria, measles, pertussis (whooping cough), pneumonia, polio, rotavirus diarrhea, rubella and tetanus. The benefits of immunization are being extended to adolescents and adults, providing protection against life-threatening diseases such as influenza, meningitis, and cancers (cervical and liver cancers).However, even now, an estimated 22 million infants are not fully immunized with routine vaccines, and more than 1.5 million children younger than 5 years old die from diseases that could be prevented by existing vaccines.The implementation of immunization programs over the past 30 years has led to remarkable achievements. The WHO European region was certified polio free in 2002 and in the past decade cases of measles in the region have been reduced by more than 90% (deaths have decreased from an estimated 535,300 in 2000 to 139,300 in 2010), which indicates the effectiveness of immunization programs. The next goal for the WHO European region is the elimination of measles and rubella by 2015. To achieve this, vaccination uptake has to reach 95% in all population groups. For this reason more needs to be done. Nearly 700,000 people in the European region do not receive basic vaccination. Furthermore, vulnerable or hard-to-reach population groups exist in all countries. Immunization has led to the control of most infectious diseases, so many parents and health professionals no longer fear those diseases. However, public confidence in vaccines is affected by groups, websites and the press. Also, in times of competing health
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priorities, the political commitment towards immunization has decreased. Immunization programs must be strengthened or the region risks the re-emergence of highly contagious diseases, causing disability and death, and placing a considerable burden on health care systems. Recent outbreaks emphasize the regional responsibility we all share to keep vaccine-preventable diseases under control.By acknowledging that every child deserves a healthy start in life, countries should increase awareness of the importance of vaccination and strengthen their immunization systems.
Pipa Efthalia, Vaccine-Preventable and Congenital Diseases Office,
HCDCP
Epidemiology of Q fever
Q fever is a zoonosis with a world-wide distribution (except New Zealand and Antarctica) caused by Coxiella burnetii, an obligatory intracellular bacterium. When in 1935 Edward Derrick named a self-limiting febrile illness of unknown etiology in nine abattoir workers in Brisbane, Australia, as Q fever (Q for query) until fuller knowledge would allow a better name, he had no idea how well the name would still fit almost 80 years later. Although we are in an era of genomics, and the availability of the complete genome of C. burnetii has increased our understanding of the disease, there are still important, unanswered questions concerning the pathogenesis of the bacterium, the clinical manifestations of the disease, the disease reservoirs, the modes of transmission and the treatment. Q fever in humans occurs with very diverse clinical manifestations: asymptomatic (50-60%), acute illness (from ’flu-like illness or very severe pneumonia) to chronic disease (mainly endocarditis) [1].
Figure 1: Reported occurrence of C. burnetii for the last 10 years (adapted from European Food Safety Authority Journal 2010;8:1723)
*: Smallest administrative region or territorial unit for statistics (NUTS), data from last 10 years
The complexity of the eco-epidemiology of Q feverCoxiella burnetii is maintained in nature through complex cycles that involve a wide range of natural hosts (wild and domestic mammals, birds, reptiles and arthropods). Humans are accidental hosts. Regarding the bacterial transmission in livestock (cattle, goats and sheep), giving birth in particular plays a dominant role. Infected animals are usually asymptomatic, but miscarriages may occur. The bacteria are excreted into the environment from the secretions of infected animals (urine, feces and milk) but mainly via the products of labor and birth (more than 109 bacteria/g placenta). The airborne pathway is the main mode of transmission (inhalation of contaminated aerosols directly from the secretions of infected animals, or secondarily from the environment) [1].As C. burnetii is highly resistant to physical and chemical stress (heat, drying, pressure and disinfectants), it survives in the environment for extended time periods, constituting a source of infection for humans and animals through the transfer of the bacterium by the wind. There are reports of dispersion from stockbreeding areas to urban areas over many miles in many unexpected places [2], infecting people who had had no previous contact with infected animals.
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Although scarce, transmission can occur after consuming unpasteurized milk and its products.The role of arthropods in the transmission of Q fever in humans is debated. Although the first identified strain of C. burnetii was isolated from a tick, and the bacterium has been found in more than 40 tick species, transmission by ticks is not considered significant, at least in humans. Instead, ticks appear to play a role in enzootic transmission cycles in wild vertebrates (rodents and lagomorphs) and wild birds [3]. Infected ticks excrete large bacterial loads through feces to the skin of the animal host during feeding. In addition, transovarial and trans-stadial transmission has been observed in ticks, allowing the bacterium’s maintenance within a population of ticks.
Figure 2: Notification of reported confirmed cases of human Q fever in member states (cases per 100,000 population), 2007-2010
Sentinel surveillance system only covers 25% of the total population in Spain.In 2010, the incidence of Q fever in EU countries was 0.36 /100,000 inhabitants. France notified cases for the first time in 2010 and, together with the Netherlands and Germany, accounted for 81.3% of the total number of confirmed cases reported in 2010
Figure 3: Overall seasonal distribution of reported confirmed human cases of Q fever in reporting member states, 2010
Source: Belgium, Cyprus, Finland, France, Germany, Greece, Hungary, Ireland, Latvia, Netherlands, Portugal, Slovenia, Spain, Sweden (Ν=1.338)
In Europe, Q fever was reported for the first time in Greece during the Second World War, when the disease was named the Balkan flu (Balkan Grippe). In an epidemic in Athens in 1943, C. burnetii was isolated from a patient to guinea pig. From 1944 to 1945, epidemics were recorded in American troops that were stationed in Italy and Corsica. After the Second World War and until the 1980s, Q fever was endemic in Europe, in Portugal, Spain, southern France, Italy, Greece, Cyprus, Switzerland, southern Germany, Slovakia, Great Britain, the Balkans and the southern Soviet Union, which reported local outbreaks or sporadic cases usually associated with stockbreeding areas. The sources of infection for humans were mainly cattle and sheep, and goats in southern Europe.Epidemiological data obtained from reference laboratories and from prevalence studies carried out during the 1990s do not provide comparable results and thus do not allow generalizations (because of differences in their design and the purpose of each study, the sample size, target groups, laboratory methods, cut-off points, etc.). For the last 10 years, there have been more organized efforts of disease surveillance of both humans and animals. However, mandatory reporting of the disease has not yet been implemented in many European countries, and even in countries that include it as compulsory reportable disease, it is under-reported. The large difference observed between the actual number of infections and the number of cases reported may be explained by the large proportion of subclinical cases and the non-specific symptoms of acute disease, which are not easily diagnosed, especially in non-endemic areas (only 20% of symptomatic patients in the epidemic in the Netherlands asked for medical care). Additionally, the difficulties in laboratory diagnosis, because of the low sensitivity of the laboratory tests and the limitations in the isolation and culture of the micro-organisms (biosafety level 3 facilities), result in failure to make a definitive diagnosis of Q fever (only 39% of patients test positive in the first serum sample) [4]. It has been reported that during the outbreak in the Netherlands, each case reported as acute Q fever infection represented >12 infections with C. burnetii [5]. Data obtained from the last few years of epidemiological surveillance of the disease in humans and animals in European Union (EU) countries are shown in figures 1-5 [6].Figure 4: Α) Reported C. burnetii (Q fever)-positive cattle, sheep and goats, 2008-
2010, split by cattle, sheep and goats.Β) Occurrence of C. burnetii (Q fever) in the reporting member states in cattle,
sheep and goats, 2010Note: Data are included only for sample sizes ≥25.
Source: for cattle, Austria, Belgium, Bulgaria, Denmark, Finland, Germany, Ireland, Italy, Latvia, Poland, Slovakia, Spain and Sweden; for sheep, Austria,
Belgium, Bulgaria Finland, Germany, Greece, Italy, Romania, Slovakia, Sweden and United Kingdom; for goats, Austria, Belgium, Bulgaria Finland, Germany, Greece,
Netherlands, Slovakia, Spain and Sweden.
Adapted from EFSA, ECDC: EU Summary Report 2010, EFSA Journal 2012;10:2597
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Data for Greece and Cyprus come mainly from the Laboratory of Clinical Bacteriology, Parasitology, Zoonoses and Geographical Medicine, Medical School, University of Crete, and are discussed further elsewhere in this newsletter.Significant changes in the epidemiological pattern of the disease have been observed during the last decade, with new outbreaks and major epidemics, as a result of changes in the organizational structure and exploitation of farm animals. Intensive farming and animal transfers between farms have favored the spread among animals and the transmission to humans. In Bulgaria during the 1990s, the increased demand for goat milk products led to a three-fold increase in the number of goats with the disease, resulting in hundreds of human cases. For the last 10 years, the spread of the pathogen observed in suburban/urban areas has been attributed to the use of manure as fertilizer in nearby cities, to animal transport through densely populated urban areas and bacterial transport by the wind. In two large outbreaks in Germany in 2003 (299 patients) and 2005 (331 patients), the source of infection for the earlier outbreak was infected sheep sold at the local market, while for the second infected sheep were being kept in proximity to residential housing [7]. In 2007 in the Netherlands, the largest, in magnitude and duration, Q fever epidemic that has ever been recorded occurred. From 2007 to 2010, more than 4,000 human cases (with 14 deaths) were recorded. In late 2009, the decision was taken to slaughter all pregnant animals on infected farms. According to the health authorities, the causes were mainly the intensification of goat herds (the number had quadrupled since 1995, surpassing 350,000). Giant modern farms, in terms of technological equipment and operation, had been developed, with high concentrations of animals near populated urban areas [8]. The characterization of isolates is necessary for us to understand the varying epidemiology of Q fever in different geographical areas. Genotyping methods are very useful tools for epidemiological investigation, particularly for clarifying links regarding the source of infection, for better understanding the emerging epidemiological factors, and to a lesser extent for evaluating control measures. Assessment of discriminatory typing methods for molecular epidemiology is in progress [9–11]. Several typing methods, such as restriction endonuclease genomic DNA, pulsed-field gel electrophoresis (PFGE), gene sequencing and polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP), have been used for the characterization of C. burnetii strains. More recently, two PCR-based typing methods have been described, multilocus variable number of tandem repeats analysis (MLVA) [12,13] and multispacer sequence typing (MST) [14], that permit the typing of C. burnetii without the need for isolation of the organism. To date, MLVA and MST are considered to be the most discriminating methods for C. burnetii, allowing the identification of up to 36 distinct genotypes. Moreover, databases have been established, http://minisatellites.u-psud.fr/MLVAnet/ and http://ifr48.timone.univ-mrs.fr, for MLVA and MST, respectively. The availability of such databases allows inter-laboratory comparisons and contributes to a better understanding of the epidemiology of the disease. The bacterial molecular profiles can be used to study transmission routes, to assess sources of infection and to assess the impact of human intervention, such as vaccination and the use of antibiotics, on the composition of the bacterial populations.
References
1.Maurin M, Raoult D. Q fever. Clin Microbiol Rev 1999;12:518-553.
2.Tissot-Dupont H, Amadei MA, Nezri M, Raoult D. Wind in November, Q fever in December. Emerg Infect Dis 2004;10:1264-1269.
3.Beaman MH, Hung J. Pericarditis associated with tick-borne Q fever. Aust NZJ Med 1989;19:254-256.
4.Fournier PE, Raoult D. Comparison of PCR and serology assays for early diagnosis of acute Q fever. J Clin Microbiol 2003;41:5094-5098.
5.van der Hoek W, Hogema BM, Dijkstra F, et al. Relation between Q fever notifications and Coxiella burnetii infections during the 2009 outbreak in The Netherlands. Euro Surveill 2012;17:20058.
6. Sidi-Boumedine K, Rousset E, Henning K, et al. Development of Harmonised Schemes for the Monitoring and Reporting of Q Fever in Animals in the European Union. EFSA Scientific Report on Question No EFSA-Q-2009-00511, 2010. Pp 48. Available from http://www.efsa.europa.eu/en/supporting/doc/48e.pdf
7.Gilsdorf A, Kroh C, Grimm S,. Large Q fever outbreak due to sheep farming near residential areas, Germany, 2005. Epidemiol Infect 2008;136:1084-1087.
8.van der Hoek W, Morroy G, Renders NH, et al. Epidemic Q fever in humans in the Netherlands. Adv Exp Med Biol 2012;984:329-364.
9. Chmielewski et al., 2009
10. Klaassen et al., 2009
11. Sidi-Boumdedine et al., 2009
12. Arricau-Bouvery et al., 2006
13. Svraka et al., 2006
14. Glazunova et al., 2005
15. European Food Safety Authority Journal 2010;8:1723
16. EU Summary Report 2010, EFSA Journal 2012;10:2597
Α. Psaroulaki, Assistant Professor, Medical School, University of Crete
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Diagnosis of Coxiella burnetii infection
Collection and storage of specimensCoxiella burnetii virulence is particularly high, and numerous cases of laboratory-acquired infections have been reported. Thus, in biohazard conditions (biosafety level 3), potentially C. burnetii-infected biological samples should be handled and processed only by experienced laboratory technicians. The same precautions should be taken for the handling of cell cultures and infected animals. Several types of human sample are suitable for the detection of C. burnetii, but their availability depends on clinical presentation. All samples, excluding whole blood, which should be kept at 4°C, should be stored at -80°C and should be forwarded on dry ice to the diagnostic laboratory [1].
Direct detection
CultureCoxiella burnetii isolation from biological samples is carried out on HEL cells using the shell vial centrifugation technique. Cell monolayers in shell vials are inoculated with the clinical specimen. The inoculated monolayers are then centrifuged and incubated. Coxiella burnetii is usually observed by microscopic examination of cell monolayers after Gimenez or immunofluorescence staining [1].
ImmunodetectionDetection of the organism in tissues is especially helpful in patients who are undergoing treatment for chronic Q fever. Samples can be tested fresh or after formalin fixation and paraffin embedding. Immunodetectionis carried out using immunoperoxidase techniques or immunofluorescence with polyclonal or monoclonal antibodies. Only this last technique can be used on paraffin-embedded samples [1]. Recently, Lepidi et al. proposed a new method, called autoimmunohistochemistry, for the detection of C. burnetii endocarditis [2].
Molecular biologyMolecular assays have been developed to detect C. burnetii DNA in cell cultures and clinical samples such as biopsy tissues (e.g. cardiac valves), cerebrospinal fluid, whole blood and serum [3]. They are applicable even in paraffin-embedded tissues. The major advantage of these techniques is the high specificity for diagnosing infections before seroconversion. Thus they are informative in patients with suspected acute infection when antibody titers are undetectable or very low [4].There are several polymerase chain reaction (PCR)-based diagnostic assays that are used only in reference laboratories. These assays include conventional PCR, nested PCR and real-time PCR. Various genes have been used. Light-cycler nested PCR (LCN-PCR) has been adapted for the diagnosis of both acute and chronic Q fever [5].
Indirect detection
Serology Because clinical diagnosis is difficult in most instances, the diagnosis of Q fever relies upon serology, which allows for differentiation between acute and chronic cases. A variety of serological techniques are available, but the most commonly used are complement fixation (CFT), indirect immunofluorescence antibody (IFA), enzyme-linked immunosorbent serologic assay (ELISA) and micro-agglutination [3]. ELISA has a high sensitivity and a good specificity. CFT is specific but less sensitive than the ELISA or IFA. The IFA test has become the reference technique: it is simple and accurate [6].
Acute Q feverSeroconversion or a four-fold increase in antibody titers, or a single serum sample exceeding the proposed cut-off values, is considered diagnostic of acute Q fever. Anti-phase II titers of IgM 1:50 or more and IgG 1:200 or more indicate recent infection [7]. In Greece, because of the high endemicity of the acute form of the disease, cut-off values have been estimated as follows: IgM ≥1:400 and IgG ≥1:960 [8]. IgM antibodies are the first to be detected, 1–4 weeks after the onset of symptoms. Their levels peak after 4-8 weeks and then gradually decrease, but are still detected 12 weeks after disease onset [9]. Anti-phase II IgG antibodies follow the appearance of IgM: their levels peak about 8 weeks after the onset of symptoms and they can still be detected after several years. On the other hand, anti-phase I IgG antibodies develop only very slowly and remain at lower titers compared with anti-II IgG [1].
Chronic Q feverWith chronic Q fever, where there is a persistence of organisms, anti-I and anti-II IgG titers may both be high. The persistence of high levels of anti-I IgG (1/800) 6 months after acute Q fever, or the reappearance of antibodies, indicates chronic infection [9]. Moreover, the presence of IgA antibody to phase I antigen (1/50) is usually associated with chronic infection [1]. According to the modified Duke’s criteria for the diagnosis of endocarditis, an anti-phase I IgG titer more than 1:800 is now considered to be a major criterion (Table 2). Recent studies suggest that the cut-off values should be placed higher in order to increase the sensitivity of the method. The proposed cut-offs for the diagnosis of C. burnetii endocarditis are higher. Specifically, a titer of anti-I IgG ≥6400 should be considered as a major criterion for endocarditis diagnosis, and a titer of anti-I IgG ≥800 and <6400 as a minor criterion. The same cut-off values are proposed as criteria for the diagnosis of Q fever vascular infections, whereas for other clinical aspects of chronic Q fever there are no specific cut-off values [10].
There can be cross-reactions between C. burnetii and Legionella pneumophila, Legionella micdadei and Bartonella quintanna/henselae, which should always be considered in the differential diagnosis [9].
Other diagnostic proceduresCurrently, IFA is the reference technique for Q-fever diagnosis, but it is poorly reproducible because of manual processing and subjective reading of the slides. InoDiag (La Ciotat, France) has recently developed a new assay for primary serologic screening for atypical pneumonia and culture-negative endocarditis, i.e. multiplexed automated corpuscular antigenic microarray (MACAM). This technique is efficient for acute and chronic Q fever. MACAM has been compared with IFA and ELISA techniques and showed consistent results [11,12].
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References
1. Angelakis E, Raoult D. Q Fever. Vet Microbiol 2010;140:297-309.
2. Lepidi H, et al. Autoimmunohistochemistry. A new method for the histologic diagnosis of infective endocarditis. J Infect Dis 2006;193:1711–1717.3. Tissot-Dupont H, Raoult D. Clinical aspects, diagnosis, and treatment of Q Fever. In: D Raoult, P Parola eds. Rickettsial Diseases. UK: Informa Healthcare, 2007; pp 291-301. 4. Fournier PE, Raoult D. Comparison of PCR and serology assays for early diagnosis of acute Q fever. J Clin Microbiol 2003;41:5094-5098. 5. Fenollar F, Raoult D. Molecular diagnosis of bloodstream infections caused by non-cultivable bacteria. Int J Antimicrob Agents 2007;30(Suppl 1):S7–S15.6. Maurin M, Raoult D. Q fever. Clin Microbiol Rev 1999;12:518–553. 7. Dupont HT, Thirion X, Raoult D. Q fever serology: cutoff determination for microimmunofluorescence. Clin Diagn Lab Immunol 1994;1:189-196.
8. Tselentis Y, et al. Q fever in the Greek Island of Crete: epidemiologic, clinical, and therapeutic data from 98 cases. Clin Infect Dis 1995;20:1311-1316.
9. Gikas A, Kokkini S, Tsioutis C. Q fever: Clinical manifestations and treatment. Expert Rev. Anti Infect Ther 2010;8:10. Raoult D. Chronic Q fever: expert opinion versus literature analysis and consensus. J Infect 2012;65:102-108. 11. Gouriet F, et al. Comparison of the new InoDiag automated fluorescence multiplexed antigen microarray to the reference technique in the serodiagnosis of atypical bacterial pneumonia. Clin Microbiol Infect 2008;14:1119–1127.12. Gouriet F, et al. Multiplexed whole bacterial antigen microarray, a new format for the automation of serodiagnosis: the culture-negative endocarditis paradigm. Clin Microbiol Infect 2008;14:1112–1118.
Meropi Gika, Resident Microbiologist, GHA ‘Evangelismos’
Q-fever: control methods and vaccination
Infection of humans and animals with Coxiella burnetii should be diagnosed early and treated immediately, in order to prevent the development of chronic infections and secondary adverse events.In humans, the recommendation is to avoid animal exposure and to use gloves and masks when in direct contact with animals or their byproducts. In the USA, post-exposure prophylaxis guidelines for the general population are 100 mg doxycycline (or 500 mg tetracycline 2×/day for 5 days), started 8–12 days post-exposure. This recommendation is not applicable to pregnant women, for whom the use of cotrimoxazole is suggested [1]. On the other hand, it is crucial to prevent the infection of animals within herds. Measures have been recommended by a number of investigators in order to prevent animal infestation and the subsequent spread of C. burnetii to humans.In France, for instance, when Q fever is identified on a farm where cheese is produced, the milk of all female animals that have aborted is discarded. If Q fever is diagnosed in a farm where fresh milk is the main product, the sale of milk from this farm is restricted for a whole year after the diagnosis has been established. Arricau-Bouvery et al. [2] recommend decontamination of feces with calcium cyanide, while in the USA the use of 2% formaldehyde, 1% lysol, 5% hydrogen peroxide, 70% ethanol or 5% chloroform is suggested for the disinfection of surfaces.Leakages of contaminated material in animal facilities should be dealt with immediately, using hypochlorite, 5% peroxide or phenol-based solutions. Current knowledge, however, suggests that disinfection of large surfaces is impossible.In the Netherlands, the scattering of manure from herds is forbidden for at least 90 days after any suspicion of infection. Manure should be processed with asbestos or calcium cyanide and it should be scattered on a day with no wind [3].
The vaccineImmunization is the most reasonable strategy for the prevention of Q fever, in animals as well as humans who are at risk.Many vaccines have been developed against animal Q fever, the most reliable being those derived from phase I-inactivated bacteria. The inactivated phase I vaccine is appropriate for the immunization of ruminants (Coxevac, CEVA, Hungary). This vaccine is highly purified and consists of entire cells, produced from phase I of the ‘Nine Mile’ C. burnetii strain. Theoretically, it can prevent abortions in animals. It is more than evident that the use of the vaccine results in control of the disease and its environmental spread. Therefore, the risk of human contamination becomes smaller.The efficacy of animal vaccination has been studied by numerous researchers. In Slovakia, a large-scale bovine vaccination program was implemented for periods of 10 years (the decades of 1970 and 1980). This, in addition to the improved veterinary controls during animal transportation within the country, resulted in a reduction of Q fever in both humans and animals [4].Recently, on the occasion of a large outbreak of animal and human Q fever, an extensive immunization program for sheep and goat farms and the control of manure processing was carried out in Holland. The efficacy of the vaccine was not proven in goats, as the expected actions of the vaccine (reducing abortions and bacterial shedding from animals infected after their vaccination) were not seen. It is still not clear if the shedding of bacteria from animals to the environment is controlled or even reduced after vaccination, proving that epidemic control is difficult and is affected by the prolonged existence of bacteria in the environment and the possible role of other animal species, apart from small ruminants, in the recurrence of an epidemic [5].The study by De Cremoux et al. [6] proved that vaccination of infected goat herds reduces clinical symptoms and the overall vaginal shedding of C. burnetii, and is more efficient in young and vulnerable animals. On the other hand, the duration of protection has not yet been
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sufficiently studied. Several authors believe that it is preferable to select seronegative herds or young animals for immunization and to continue vaccinating young animals for several years [7].
In sheep and goats, vaccination cannot be used for controlling infection in already infected animals, and it is more effective in animals that have not given birth. The phase I inactive vaccine (Coxevac, CEVA Sante Animale, Libourne, France) provides protection against abortions and has been proven to reduce bacterial dissemination by helping decrease the bacterial load in vaginal mucous, feces and, especially, milk.In milk-producing bovines, vaccination significantly reduces the possibility of dissemination by an animal, but has no effect on the bacterial load. Vaccination of previously infected animals or non-infected animals during pregnancy has proved inefficient [8].New recombinant vaccines have been developed. Most of these have an antigen-producing action but are not protective [9]. Vaccination of humans against Q fever could be efficient in some parts of the world. In endemic areas, it could be beneficial for the professionally exposed population, including workers in abattoirs and the milk industry.It is also possible that professionals who are not exposed are at risk of developing the chronic form of the disease and could benefit from the use of such a vaccine. This group includes individuals with valvular disease, vascular aneurysms or even immunocompromised patients with prosthetics [10].A variety of human vaccines has been produced in different parts of the world but has never been put to use. For the time being, the only one that is used is the inactivated phase I vaccine, which was derived from the protective antigen of purified, intact phase I lipopolysaccharide (LPS) of the Henzerling strain, after its inactivation with formaldehyde. This vaccine was approved by the Australian authorities in 1989 (Qvax, CSL Limited, Parkville, Victoria, Australia) A series of trials has already proven its efficacy, although immunization is contraindicated in individuals who display seroconversion or have previously been exposed to C. burnetii. Adverse events are frequent and important, thus a pre-immunization trial dose is suggested, making this preventive measure expensive and time consuming. A single dose of the 30-μg vaccine provides immunity after 10–15 days, which lasts for at least 5 years. Gefenaite et al. [11] have published a meta-analysis of previous studies concerning the efficacy of the Qvax vaccine. All trials included in the meta-analysis conclude that vaccination is beneficial and protective (median efficacy after the collection of initial data 97%, confidence interval 94-99%).
References
1. Moodie CH, Thompson HA, Meltzer MI, Swerdlow DL. Prophylaxis after exposure to Coxiella burnetii. Emerg Infect Dis 2008;14:1558–1566.
2. Arricau-Bouvery N, Souriau A, Moutoussamy A, et al. Study of Coxiella burnetii excretion in an experimental goat model and decontamination of dung with calcium Cyanamid. Rencon Rech Rum 2001;8:153–156.
3. Schimmer B, Morroy G, Dijkstra F, et al. Large ongoing Q fever outbreak in the south of the Netherlands, 2008. Euro Surveill 2008;13:article 2.
4. Kovacova E, Kazar J. Q fever—still a query and underestimated infectious disease. Acta Virol 2002;46:193–210.
5. European Food Safety Authority (EFSA). Panel on Animal Health and Welfare (AHAW); Scientific Opinion on Q Fever. EFSA J 2010;8:1595. doi:10.2903/j.efsa.2010.1595. Available online www.efsa.europa.eu
6. De Cremoux R, Rousset E, Touratier A, et al. Assessment of vaccination by a phase I Coxiella burnetii inactivated vaccine in goat herds in clinical Q fever situation. In Proceedings of the 6th International Meeting on Rickettsiae and Rickettsial diseases, Heraklion, Crete, Greece, June 2011.
7. Krauss H. Clinical aspects and prevention of Q fever in animals. Eur J Epidemiol 1989;5:454–455.
8. Guatteo R, Joly A, Rodolakis A, et al. Prevention de l’excretion de Coxiella burnetii a l’aide d’un vaccin dit phase I (Coxevac en troupeaux bovines laitiers infectes). Rencon Rech Rum 2008;15:59–62.
9. Waag DM. Coxiella burnetii: host and bacterial responses to infection. Vaccine 2007;25:7288–7295,.
10. Maurin M, Raoult D. Q fever. Clin Microbiol Rev 1999;12:518–553
11. Gefenaite G, Munster JM, van Houdt R, Hak E. Effectiveness of the Q fever vaccine: a meta-analysis. Vaccine 2011;29:395–398.
Achilleas Gikas, Professor of Infectious Diseases, Medical School, University of Crete
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The role of the Laboratory of Clinical Bacteriology, Parasitology, Zoonoses and
Geographical Medicine, Medical School, University of Crete, in Q fever research and control in
Greece
The Laboratory of Clinical Bacteriology, Parasitology, Zoonoses and Geographical Medicine, Medical School, University of Crete, was founded in 1985. The laboratory joined the collaborating laboratories of the World Health Organization (WHO) Collaborating Center for Research and Training in Mediterranean Zoonoses (MZCC/WHO) and was a national reference center under the direction of Professor G. Tselendi.Among other zoonotic pathogens, Rickettsiae and Coxiella burnetii (Q fever) were a major target of the laboratory’s work, and thus the foundation of the first research group for C. burnetii was established by the newly integrated laboratory along with the experience gained at the Laboratory of Physiology of the University of Athens and the Pasteur Institute of Athens. Their experience was further broadened with the co-operation and training of personnel from the Institute Pasteur in Paris (Professor Etlinger) and the University of Marseille (Unité des Rickettsies, Professor Raoult).Q fever and C. burnetii was a little-studied subject during the first years of the research activity of the laboratory. Given the uniqueness of the bacterium (obligate intracellular survival and difficulties in culturing), the problems of diagnosis (laboratory diagnosis of chronic–acute disease), and also in monitoring and adjusting treatment, part of the research activity focused on improving the diagnostic methods and control of chemotherapy treatment. New methods of rapid laboratory diagnosis were developed and implemented: isolation (shell-vial technique), cell culture, and detection and typing using molecular biology techniques (new methods that began to be developed and evolve during the 1990s). As conventional methods of calculating the MIC of antibiotics cannot be applied to C. burnetii, a new method of determining the sensitivity of antibiotics in cell cultures was developed and applied.A large number of scientists from various specialties, from Greece, Cyprus, Albania, Serbia, Syria and other countries in the Balkans and Eastern Mediterranean, were trained in these methods.The research objectives of the laboratory were as follows.
• Study of the various vectors of the disease. Thereafter the focus was on the transmission dynamics of C. burnetii in space and time using genotypically characterized bacterial strains from their hosts (hosts-mammals/arthropods-vectors). Continuous monitoring: investigation of the prevalence of the disease in populations of all links in the epidemiological chain (humans, livestock, wild mammals and arthropods).
• Investigation of the genetic diversity of strains of the bacterium and determination of the relationship of different genotypes with clinical manifestations of the disease.
• Exploration of the complex system of transmission of C. burnetii, (frequency-dependent/density-dependent mode of transmission) and the connection and interaction of the suburban transmission cycle with the cycle in wildlife.
• Exploration of the role and significance of ticks in the epidemiological chain (frequency-dependent mode of transmission) and evolution of the pathogen by changing its mode of transmission (density-dependent mode of transmission)
• Samples for these studies were: human samples from patients and the healthy general population (>10,000 samples); samples from livestock (goats, sheep, cattle, n >3000), wild mammals (rodents, mouflons, foxes, hares, hedgehogs, n >1500), migratory and endemic birds (n = 600 from 50 different species) and arthropods (ticks, fleas, >5000) (Table 1). The tools used for the investigation were rapid isolation techniques and culture (shell vials/cell cultures), methods of molecular diagnostics and molecular epidemiology and geoinformatic methodologies [geographical information system (GIS)], etc. The results were the first produced in Greece and Cyprus and contributed to our understanding of the clinical presentation and epidemiology of the disease. Tables 1 list the titles of the scientific papers and some of the results of the studies.
Table 1: Epidemiological studies of Q fever in Greece and Cyprus
GREECE CYPRUS
HUMAN POPULATION
Patients
1989-1993Retrospective clinical-
epidemiological study (Crete) 1.298 patients with possible
Q fever98 confirmed cases. The
clinical manifestations and risk factors were described.
Tselentis Y, 1995
1989 -19963.466 patients with probable
acute infection274 cases with serologic evidence of acute infection. A total of 138 cases fulfilled the serological and clinical criteria.
Spyridaki I, 1998 &
Voloudaki A 2000 & Gikas A, 2001
Data (10 years) from Greece and Cyprus
67 patients with chronic Q fever infection in Greece and Cyprus
Unpublished data
Healthy population
1987 - 1985 Seroepidemiological study in
two Cretan villagesSeroprevalence of 38.1% and 13.5% respectively (random
sampling)
1996-1999 Seroepidimiological study
using representative, random sampling in Cyprus (n=583)
Seroprevalence: 52,7% (random sampling in general
population) (IgG cutoff >1/60)
Incidence of the disease:1.2 cases/year/100.000
population
Loukaides 2006 & Psaroulaki 2006
Forage area residents (Crete) 1985 -1987 : 231 residents
(50 families) 34,6% seropositivity 1998: 238 residents (84 families)
42% seropositivity
Antoniou M, 2002
2008 Crete: healthy blood donors, random sampling (n=493)
Seroprevalence:48.7% (indicating high exposure of the population irrespective of
occupation or residency)
Vranakis 2012
2008 Crete: Forage area residents
(n=225) 62.2% seropositivity
2004-2007 Cyprus
Random sampling in general population (n=375)
Seroprevalence:52.7%
Vranakis 2012
ANIMAL POPULATION
2008Ranching region (Crete)
(n=244)Seroprevalence in sheep: 67%
Unpublished data
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1996-1999 Cyprus: random sampling representative of the total population (n=974 sheep,
goats and cattle) Seropositivity: 48.2 %
(goats), 18,9 % (sheep), 24 %(cattle) (IgG cutoff >1/60). The analysis of
possible risk factors for the disease transmission revealed three important risk factors:
occupation, possession of goats and sheep, use of
manure in gardens.
Psaroulaki A, 2006
Cattle, sheeps, goats, dogs
2004-2007n= 1017 (346 goats, 333
sheep, 338 cattle) Total seropositivity:69% Statistically significant
difference between the little ruminants (goats and sheep)
in comparison with cattle (p‹0,001)
Cattle:40%, goats:84%, sheeps:81%
139 dogs were also controlled (seropositivity:3,59%)
Unpublished data
Wild forest animals
2004-2007 C. burnetii was detected using
PCR methods in: Mouflons: 25% (n=74) Foxes: 30% (n= 32) Hares: 48% (n=247)
557 species of migratory/endemic birds were
controlled. C. burnetii was detected in 51
species of birds.
Ioannou I 2009 & Ioannou I 2011
2000-2003 622 rodents
Collection from 51 different areas of Cyprus
Seropositivity:12,8%
Psaroulaki A, 2010
TICKS
2003-2005 Cefallonia Sampling: 1848 ticks from sheep, goats, cattle, horses
and dogs
Psaroulaki A 2006
1996-1999 Genotypic detection of C.
burnetii in 10% of the ticks (n=141) -isolation of the
bacterium
Spyridaki I, 2002
2004-2007 n=1761 ticks (7 species) from cattle, goats, sheep and dogs
n=1135 ticks (10 species) from wild animals
Genotypic detection of C. burnetii in 3 tick species
which infested goats, sheep, cattle as well as 5 tick species
which infested foxes, hares and mouflons
Use of the large volume of data generated, as well as the genotyping/sequencing of C. burnetii isolates from the laboratory microbe collection, is currently under way. As well as geoinformatic tools, data analysis is being performed using methodologies of community epidemiology, the genetic structure of populations, the evolutionary biology of the pathogen, and mathematical models. The objectives of this analysis are the confirmation, or not, of the hypothesis of the transition of the pathogen (jumping) from frequency-dependent (ticks) to density-dependent transmission, the development of mathematical models of pathogen transmission and simulation with existing data in space and time, and the association of different genotypes with the clinical expression of the disease Q (especially endocarditis), etc.By applying proteomic techniques (since 2003), in collaboration with the Department of Chemistry, University of Crete, and Ghent University (Belgium), we have investigated the following.
• The ability of the bacterium to cause chronic disease. The results of a series of studies have revealed that between acute and persistent infection there are major protein rearrangements, both in the bacterium and within the eukaryotic cell, indicating that C. burnetii orchestrates a huge number of different bacterial and eukaryotic host cell processes to survive within the cell. The pathways that have been identified have not only added to our knowledge of the pathogenesis of the bacterium but are also ideal targets for the development of new drugs.
• The effect of the bacterium in the host cell in favor of its proliferation. We have developed a C. burnetii-secreted protein isolation protocol from the cytoplasm of the infected eukaryotic host cell. By using proteomic tools we have identified secreted bacterial effector proteins (molecular effectors) that could be ideal drug targets for the collapse of the bacterium’s parasitism.
• Potential diagnostic biomarkers capable of distinguishing the acute from the chronic form of the disease, contributing to the diagnosis of Q fever.
• The existence of possible antibiotic resistance mechanisms in C. burnetii for antibiotics used for treatment. The results of these studies have revealed previously unknown biochemical pathways in C. burnetii utilized by the bacterium, leading to resistance to quinolones and doxycycline.
Over the years the laboratory has developed a network of collaboration with hospitals in Greece and Cyprus. Thousands of human samples have been tested and dozens of patients with chronic Q fever monitored.The conclusions from the investigations mentioned above have been published in 11 research articles.The data obtained from proteomic studies at the level of diagnostic markers are currently being evaluated against sera from patients with Q fever from the collection of the laboratory, to develop, initially, in-house diagnostic tests. Protein-potential drug targets that have already been identified are being evaluated for possible use in the treatment of Q fever. The goal is to evaluate (in silico and in vitro) the effectiveness of these substances in the collapse of the parasitism of the bacterium. At the level of resistance mechanisms to the antibiotics used for treatment, the ultimate objective of the laboratory is to investigate the evolutionary biology of bacterial resistance through step-mutations.The laboratory has drafted and implemented seven funded research projects (European
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competitive, transnational collaborations and national funding). We have developed partnerships with the following scientific institutions: Department of Chemistry, University of Crete; Athens Academy; Faculté de Médecine University of Marseille; Ghent University (Department of Medical Protein Research and Department of Biochemistry); Laboratory of Intracellular Parasites, Rocky Mountain Laboratories (Coxiella Pathogenesis Section); Institute for Medical Research, Belgrade; and others (Veterinary Services and the Cyprus Game Fund, etc.).The laboratory research activity concerning Q fever is reflected in 33 publications (Table 2) and 10 theses, either already completed or currently in progress. Three members of the research team are ranked as PubMed experts for C. burnetii and Q fever.
Tselentis Yannis, Professor, Psaroulaki Αnna, Assistant Professor Medical School, University of Crete
Clinical manifestations and treatment of Q fever: what’s new?
Q fever may manifest as acute Q fever or chronic Q fever with long-term sequelae [1]. Up to 60% of infected patients develop no symptoms [2]. Major differences in the manifestations of Q fever occur in various countries. In Crete (Greece) and Maritime Canada, pneumonia predominates; in Australia, fever with no apparent localization of infection predominates; and in France, southern Spain and the Canary Islands, fever and hepatitis predominate [1,3]. Acute Q fever (incubation time 2-5 weeks) usually develops as a non-specific febrile illness, pneumonitis or hepatitis [2]. Derrick’s description of the illness often included a severe headache behind the eyes [1]. Typically a high fever of 39-40°C reaches a plateau in 2-4 days and then, after 5-14 days, the temperature returns to normal [4]. Atypical pneumonia is one of the most classic forms of Q fever. However, hepatitis is the most common form of infection by Coxiella burnetii and may be expressed as infectious-like hepatitis or FUO with characteristic hepatic granulomas on liver biopsy [4]. Less common manifestations of acute Q fever include myocarditis, pericarditis, meningoencephalitis, skin rash and more (Table 1). There is a male predominance (a male/female ratio of 2.45), with a range of duration of symptoms prior to diagnosis from 1 to 14 months [2]. Chronic Q fever manifests as bacterial culture-negative endocarditis in up to 75% of cases, many months to years after the initial infection. Predisposing conditions include pre-existing valvular disease, vascular abnormalities and immunosuppression [2]. Vegetations are usually absent or small. Mitral and aortic valves are affected most, although recently a world-wide increase in prosthetic valve endocarditis has been reported [4]. Patients with endocarditis may have received multiple antibiotics prior to valve replacement, making isolation of the organism from the infected tissue improbable [2]. In addition, chronic Q fever may appear as a vascular infection of an aortic aneurysm or a vascular graft, osteo-articular infection, chronic hepatitis, chronic pulmonary lesion and chronic fatigue syndrome [2] (Table 1). Q fever in pregnancy may manifest as abortion, premature birth or neonatal death. Finally the clinical presentation of Q fever in children is similar to that in adults and some deaths have been reported. Recently special attention has been given to the relationship between C. burnetii and chronic fatigue syndrome; 28% of Australian cases met the CDC criteria of chronic fatigue syndrome [1].
Treatment
Acute Q feverFor acute infection, the recommended treatment is doxycycline (100 mg orally BID) for 14 days, starting antibiotics until day 3 of fever, increasing the efficiency of the therapy [5]. In untreated patients the mortality rate remains low, except in cases complicated with endocarditis [6]. For patients with a valvulopathy who develop acute Q fever, some clinicians recommend 1 year of doxycycline plus hydroxychloroquine to avoid the development of endocarditis as in 30% of such patients [1]. Tigecycline, a novel tetracycline derivate with bacteriostatic action, has shown important in vitro activity against C. burnetii and may represent a novel agent in the treatment of acute Q fever. Further clinical studies are needed to elucidate its role in C. burnetii infections.Minocycline, another tetracycline, at a dose of 100 mg orally BID, has been used successfully in Japan for the treatment of acute Q fever and post-Q fever chronic fatigue syndrome [7]. Minocycline could serve as an alternative to doxycycline, especially in countries such as Greece, where there is a shortage in doxycycline supplies. Macrolides such as clarithromycin (per os 500 mg BID) and fluoroquinolones (pefloxacin 400 mg/day) show some promise [1,5]. Fluoroquinolones may be considered in cases of meningoencephalitis because of their good penetration of cerebrospinal fluid [4]. Cotrimoxazole and rifampin are alternative treatment options in the case of allergy or contraindication to tetracyclines. With regard to pregnancy, treatment with cotrimoxazole 800/160 pos BID plus folinic acid 25 mg OD, until delivery, is recommended. After delivery, if serology suggests chronic infection,
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women should be treated as for chronic Q fever to prevent subsequent abortions or endocarditis. Breastfeeding is absolutely contraindicated. For children <8 years old cotrimoxazole or the newer macrolides are the drugs of choice [1], although doxycycline (100 mg/day) is not an absolute contraindication for children with Q fever [4]. Moreover, in children with severe disease or prolonged fever, the addition of corticosteroids or interferon-γ might be considered [4]. After treatment of acute Q fever, all patients should be followed up regularly at 3 and 6 months, and if titers of phase I IgG antibodies (Ab) are >1:800, polymerase chain reaction (PCR) and transesophageal echocardiography should be performed to rule out chronic infection [4].
Chronic Q feverCurrent guidelines recommend at least 18 months of doxycycline 100 mg orally BID plus hydroxychlorochine 600 mg orally once a day. Hydroxychlorochine acts by raising the phagolysosomal pH, thus increasing the efficacy of doxycycline. Serum levels of hydroxychlorochine of 0.8-1.2 mg/L and doxycycline of more than 5 mg/L have resulted in better clinical outcomes [1]. For patients with serum levels of doxycycline <5 mg/L (slow responders), adjustment of the doxycycline dosage to 300-400 mg/day may be crucial for a positive treatment outcome [8].A clinical cure is defined as a decrease of phase I IgG and IgA Ab titers to <200 [1]. Rolain et al. observed an interpersonal variability of the kinetics of Ab titers, with slower decrease rates in some patients. Chronic Q fever needs a life-time of regular following up.Valve replacement and urgent surgical intervention may be required in the case of hemodynamic problems. The treatment of vascular Q fever does not rely on definitive guidelines. Most patients have received various combinations of doxycycline with hydroxychlorochine, fluoroquinolones or rifampin for a long period of time, sometimes for more than 3 years (with an average of 23 months), combined, where feasible, with surgical repair of the infected vessel. The combination of doxycycline with hydroxychlorochine may offer a small advantage. Major complications are frequent (23%), occurring mostly in conservatively treated patients. For post-exposure prophylaxis (PEP) the recommended regimens consist of 100 mg doxycycline BID or 500 mg tetracycline BID for 5 days started 8-12 days post-exposure. According to CDC, in immunocompromised patients and pregnant women PEP must be considered when the probability of exposure is above the population-specific threshold level.
Table 1: Clinical syndromes associated with Q fever
ACUTE DISEASE CHRONIC DISEASE
Common presentation Common presentation
’Flu-like illness, pneumonia, hepatitis Endocarditis
Less common presentations Less common presentations
Neurological: meningoencephalitis, meningitis, encephalitis, cerebellitis, neuritis, Guillain-Barre syndrome, myelitis, peripheral neuropathy, extrapyramidal disease
Vascular infections including vascular grafts infections
Gastrointestinal: gastroenteritis, pancreatitis, splenic rupture, mesenteric panniculitis, acalculus cholecystitis
Osteo-articular infections: osteomyelitis, osteoarthritis
Cardiac: myocarditis, pericarditis Granulomatous hepatitis, chronic hepatitis
Genital: orchitis, priapism, epididymitis Chronic pulmonary infections
Hematological: hemophagocytosis, uremic syndrome, anemia (hypoplastic or hemolytic), rhabdomyolisis, bone marrow necrosis
Infections during pregnancy
Endocrine: thyroiditis, inappropriate secretion of antidiuretic hormone
Long-term sequelae: chronic fatigue syndrome, cardiovascular disease, spontaneous abortion, prematurity
Cutaneous: maculopapular or pruritic rash, erythema nodosum
Renal: glomerulonephritis
Other: ARDS, lymphadenopathy
References
1. Parker NR, Barralet JH. Q fever. Lancet 2006;367:679-688.
2. Karakousis PC, Trucksis M. Chronic Q fever in the United States. J Clin Microb 2006;44:2283-2287.
3. Marrie TJ, Campbell N. Q fever update, Maritime, Canada. Emerg Infect Dis 2008;14:67-69.
4. Angelakis E, Raoult D. Q Fever. Vet Microbiol 2010;140:297-309.
5. Gikas A, Kofteridis DP. Newer macrolides as empiric treatment for acute Q fever infection. Antimicrob Agents Chemother 2001;45:3644-3646.
6. Scott JW, Baddour LM. Q fever endocarditis: the Mayo Clinic Experience. Am J Med Sci 2008;336:53-57.
7. Ohguchi H, Hirabayashi Y. Q fever with clinical features resembling systemic lupus erythematosus. Intern Med 2006;45:323-326.
8. Lecaillet A, Mallet MN. Therapeutic impact of the correlation of oxycycline serum concentrations and the decline of phase I antibodies in Q fever endocarditis. J Antimicrob Chemother 2009;63:771-774.
Stamatios Velakoulis, Aggelos Stefos, Nikolaos V. Sipsas,Infectious Diseases Unit and Department of Internal Medicine, GHA
‘Laikon’ and National and Kapodistrian University of Athens
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Invest in the Future: Defeat Malaria, World Malaria Day, 25 April 2013
Malaria is an infectious disease caused by the parasite “Plasmodium” and is mainly transmitted to humans through the bite of the infected Anopheles mosquito. There are five Plasmodium species that affect humans: Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae και Plasmodium knowlesi.Malaria due to Plasmodium falciparum is the most deadly form and it predominates in Africa; Plasmodium vivax is less dangerous, but more widespread, and the other three species are found much less frequently.
Global malaria epidemiological facts Malaria is endemic in 104 countries (2012 data) around the world, mainly in sub-Saharan Africa, Asia and Latin America (map). According to the latest estimates, in 2010 there were about 219 million malaria cases and an estimated 660 000 malaria deaths. Most deaths occur among children under five years of age living in Africa, where a child dies every minute from malaria. Globally, an estimated 3.3 billion people -half the world’s population- are at risk of malaria (2011 data), with populations living in sub-Saharan Africa having the highest risk of acquiring malaria: approximately 80% of cases and 90% of deaths are estimated occur in the WHO African Region. Malaria is strongly associated with poverty: people living in the poorest countries are the more vulnerable to malaria. Worldwide is the leading cause of death in young children, especially those living in remote areas with limited access to health services.Malaria is a preventable and treatable disease, provided the currently recommended interventions are properly implemented. Increased prevention and control measures have led to a reduction in malaria mortality rates by more than 25% globally since 2000 and by 33% in the WHO African Region. WHO goals and objectives for malaria control and elimination include: to reduce global malaria cases by 75% from 2000 levels by end 2015, to reduce global malaria deaths to near zero by end 2015, to eliminate malaria by the end of 2015 in 10 new countries (since 2008) and in the WHO European Region (source: World malaria report 2012, WHO).
Picture 1: Malaria in the World, WHO, 2011
Source: World Health Organisation, 2011, Map: WHO Global Malaria Programme
Malaria Epidemiology in GreeceGreece has been malaria-free since 1974, after the implementation of an intensive eradication program (1946-1960). Until 2010, approximately 20-50 cases were reported annually to the Hellenic CDC, the majority of which were imported, travel-related. Sporadic malaria cases
without reported travel history were recorded in 1991, 1999, 2000, 2009 and 2010. In 2011 malaria re-emerged in the country, with a cluster of 36 locally acquired P.vivax malaria cases recorded in the Municipality of Evrotas (Lakonia District). In 2012, 18 locally acquired P.vivax malaria cases were recorded nationwide, 10 of which in the Municipality of Evrotas. Most affected areas are rural, non touristic, close to wetlands, with large immigrant populations from malaria endemic countries.Hellenic CDC activities for the management of malaria - 2012 During the spring of 2012, the Hellenic CDC developed an Action Plan for the Management of Malaria, 2012-2015, where the risk assessment for re-emergence of malaria in the different areas of Greece was included. According to this Action Plan, the Hellenic CDC has performed a series of activities in 2012 to prevent the re-establishment of malaria in Greece, in collaboration with the University of Thessaly to implement the national project «Integrated Surveillance and control programme for West Nile Virus and malaria in Greece». These activities include: I. Enhancement of malaria surveillance and early detection of cases: 1) focus investigation 2) active malaria case detection in the general and the migrant population, with regular door-to-door visits (twice a month) to all immigrants houses from late September 2011 in the Municipality of Evrotas, 3) screening of migrants for malaria in affected areas and detention centers, 4) development of geographic information systems (GIS) for risk assessment.II. Enhancing laboratory diagnosis of malaria: training sessions for laboratory personnel and distribution of rapid diagnostic tests to Health Units in affected areas.III. Standardization of the malaria treatment, according to treatment guidelines developed by the Hellenic CDC. IV. Increase awareness amongst health professionals for the timely diagnosis and appropriate treatment of malaria cases.V. Communication to the public on malaria and personal protection measures against mosquitoes. VI. Communication with international public health stakeholders.VII. Vector control activities - Entomological surveillance - Training of the regional staff.
ConclusionsDespite the increased sensitivity of the malaria surveillance (active case detection, increased awareness of health professionals), in 2012 fewer locally-acquired malaria cases were recorded, compared with 2011, all over the country (57% decrease) and in Evrotas, Lakonia (72% decrease). Furthermore, active case detection in Evrotas improved significantly the timeliness of diagnosis of malaria in the area. However, the risk of malaria re-establishment in high risk areas of the country exists, since both the malaria competent vector (mosquitoes of the genus “Anopheles”) and malaria patients (especially recently arrived immigrants from endemic countries) are present. The re-establishment of malaria in the country directly depends on maintaining a high level of health care and public health services and the appropriate implementation of proper vector control programs aiming to stop the disease transmission.Therefore, the development of a national strategy and implementation of an integrated plan for the management of malaria was considered crucial, targeting simultaneously to all interventions (vector control, communication with the public and health professionals, enhanced surveillance). In this context, the National Action Plan for the Management of Malaria, 2013-2018 of the Ministry of Health is completed, aiming to maintain the malaria-free status of the country and avoid malaria re-establishment in areas where cases occurred in the past, through coordinated actions, which will be developed systematically over the following years at both national and regional level.
D. Pervanidou, A. Vakali, E. Papanikolaou, I. TerzakiOffice for Vector-borne Diseases, HCDCP
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Completion and success of the European Territorial Collaboration Program between
Greece and Bulgaria 2007-2013
The Hellenic Center for Disease Control & Prevention (HCDCP) with the Peripheral Laboratory of Public Health of East Macedonia-Thrace participated in the European Territorial Collaboration Program between Greece and Bulgaria 2007-2013. In the context of the above program, the project entitled Cross-Border Research Center Environment and Health (RCEH) has been completed.
This collaboration started in May 2011 and had the aims of creating a new laboratory for public health in the territory of Kirtzhali, Bulgaria, sharing technical knowledge and training Bulgarian scientists by the respective Greek associates in order to create a cross-border system of supervision and analysis of environmental dangers regarding public health, as well as the acquisition of new scientific equipment for the Peripheral Laboratory of Public Health of East Macedonia-Thrace
On 27 February 2013, at Kirtzhali, the official closing of the program took place. The brand new building (laboratory) that has been constructed at the center of the town, equipped with high-end equipment and a specialized work force, is now available for the people of Kirtzhali and Bulgaria in general.
Ms El. Hadjipashali, Deputy Director of the Public Health Laboratories Network (PHLN), and Ms J. Staikova, Director of the Bulgarian Public Health Laboratory, highlighted their appreciation of the successful completion of the program and pointed out that the benefits for both countries are many and of great importance, as from now on a new and close collaboration at all levels can begin, aimed at protecting public health in the area. Moreover, they both emphasized the professional and punctual completion of the program. Present at the event were representatives from the Bulgarian Ministry of Health, the medical and scientific institutes of Bulgaria, Kirtzhali’s Mayor, the Hellenic consulate, as well as the work force from the Peripheral Laboratory of Public Health of East Macedonia-Thrace, HCDCP and the European Program Office.Additionally, on 6 March 2013, at the Peripheral Laboratory of Public Health of East Macedonia-Thrace-Alexandroupoli, another official ceremony was held for the completion of RCEH in the context of the regional collaboration between Greece and Bulgaria 2007-2013. Invited to this ceremony were all the parties involved from the broader region of East Macedonian-Thrace. The event was very successful, with a high attendance. A briefing about the specific program and the work in general of the Peripheral Laboratory of Public Health of East Macedonia-Thrace took place in the presence of the Bulgarian consul.
The specific program was considered and proposed as a model of Good Corporate Practice and Collaboration. This distinction honors our country, the HCDCP and all the participants from the Peripheral Laboratory of Public Health of East Macedonia-Thrace and the European Program Office, as well as everyone else who has contributed to this program. This program is considered to be a special distinction for everyone involved.
El. Hadjipashali, Deputy Director of PHLN-HCDCP
Interview
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Future conferences and meetingApril 2013
4 APRIL 2013
Title: 28th Medical Congress of Northern Greece
Country: GreeceCity: SalonikaVenue: Hyatt Regency Hotel Phone No.: + 30 2310 460682Website: http://www.praxicon.gr/voreioelladiko.html
6-7 APRIL 2013
Title: Pathology of the Embryo
Country: GreeceCity: LarissaVenue: University Hospital of LarissaPhone No.: + 30 2413502795Website: http://allevents.in/
26-28 APRIL 2013
Title: Clinical Workshop on Updating the Clinical Management of Cardiovascular Danger Factors
Country: GreeceCity: NafplionVenue: Amalia Hotel Phone No.: +30 2107210001Website: http://www.congressworld.gr/images/afises/afisa26-04-2013.jpg
Office for Public and International relations, HCDCP
Professor Achilleas Gikas
What was your basic motivation for becoming a medical doctor?
One can decide to study medicine for several reasons. The humanitarian aspect of medicine may seem attractive to young people. It is also a matter of complex personal beliefs, morals and practical enquiries that boil down to choosing medicine, which finally becomes a way of life.
Why did you choose to specialize specifically in pathology/infectious diseases?
Largely, it’s a question of personality; specific medical specialties fit some individuals better. Personally speaking, pathology offered me a better understanding of medicine as a whole. I was blessed with being able to study under the supervision of excellent teachers who, as well as transferring scientific knowledge, also offered moral paradigms. My quest boiled down to the specialty of infectious diseases, something that fulfilled all the criteria already set by then. My acquaintance with Professor Janis Tselentis, a renowned authority in the particular field of infectious diseases and an important personality, played a crucial role in my choice. He taught and guided me to specialize further in the hospital settings of Pitie-Salpetriere, Claude-Bernard and Institute Pasteur of France, and on my course afterwards. When I returned to Greece, I was offered a position as associate professor in the Medical School of the University of Crete and my basic research was on rickettsioses and the control of nosocomial infections.
When have you experienced the deepest satisfaction in the course of your career?
A medical doctor’s life is full of scientific ‘adventures’ and disappointments but is not empty of satisfactions! Every time a period in my scientific evolution is completed, every time a research project reaches its goal or a paper is published, is a moment of satisfaction. But this only marks the setting of new targets and the circle of quest and vigilance starts over again.
Do you have any scientific plans for the immediate future, such as finishing some research or publishing a scientific paper?
Having participated in two major clinical and research projects, rickettsioses and infections in nosocomial infections, and having worked with excellent colleagues, I am satisfied by contributing to our increasing scientific knowledge, and also embracing young scientists and helping them advance their careers.
Myths and truths
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Is there any field within the infectious disease specialty that attracts you more? If yes, which one and why?
I can say rickettsioses and nosocomial infections attract me equally.
Can you make a prediction regarding infectious diseases? Do you believe that the infectious disease ‘map’ will change in the future?
Infectious disease science has played a pivotal role in the welfare of humankind. The invention of antibiotics, new diagnostic procedures, advancements in epidemiology and the public health science have offered a great deal of help to humanity. Two of the most important achievements in the 20th century have been vaccines and control over infectious diseases. Scientists’ worries about the appearance of pandemic strains of micro-organisms and exhaustion of the infection-fighting capability of antibiotics is justified, in particular the pandemic of human immunodeficiency virus (HIV), despite the stabilization of cases appearing each year, along with the geographical expansion of emerging or re-emerging diseases (e.g. malaria and West Nile fever/encephalitis) in new areas and continents, especially in Europe, causing public health alerts. The scientific community, in collaboration with state authorities and international bodies, must closely observe the evolution of infectious diseases, in order to maintain real-time surveillance and be able to respond quickly and effectively.
How do you think the public can better accept the message of prevention regarding infectious diseases, for example vaccination against seasonal influenza?
With better organization of the health services, promoting public awareness, maintaining the surveillance of the spread of infectious diseases and effectively responding with a cohesive plan and, finally, educating the public appropriately and reinforcing a sense of collaboration and trustfulness within society.
Epidemiological surveillance aims to monitor infectious diseases in order to intervene if needed. How do you believe doctors can increase their participation in the process of reporting cases of infectious disease within the national mandatory notification system?
Mandatory reporting does not fit very well with the profile of a Greek medical doctor, and a well devised plan of education, including information feedback, may establish an attitude more conducive to participating. Feedback in particular, as a reward and justification for participating, can be highly motivational.
Edited by George Dougas
Myths TruthsCoxiella burnetii survives and multiplies in macrophages via mechanisms that are used by most intracellular bacteria.
It was a general belief for many years that C. burnetii does not inhibit the fusion of phagosome with lysosome but is able to replicate in a fully formed phagolysosome in the presence of hydrolytic and other denaturing enzymes. However, recent studies indicate that the parasitophorous vacuole simply bears protein markers of a mature phagolysosome and the acidic pH found in phagolysosomes without eventually merging with the lysosome. In this way not only does the bacterium manage to ‘hide’ by macrophages, but via protein secreted in the cytoplasm of the host cell with an effector function it manipulates the host cell mechanisms to its advantage. Thus the bacterium ‘hides’ but is not ‘sleeping’; it participates in the biosynthesis and maintenance of the phagosome as long as it survives in it.
The genus Coxiella includes only one species, C. burnetii, the population of which is considered homogeneous and not heterogeneous.
Coxiella burnetii populations have a heterogeneous structure. The heterogeneity of the bacterium was initially investigated by polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) and more recently strains of acute and chronic disease have been genotyped by multilocus variable number of tandem repeats analysis (MLVA) and MLST methods, which have confirmed the polymorphism of isolates. It is generally believed that these distinct genotypes of C. burnetii are associated with the various clinical forms of Q fever. Additionally, the acute and chronic forms of Q fever have been associated with the presence of plasmids that are constantly maintained in strains of C. burnetii (e.g. acute disease strains carrying plasmid QpH1 and chronic disease-associated strains carrying plasmid QpR5). Despite the fact that the DNA of these plasmids has been fully sequenced, the pathways of gene expression have not been studied to such an extent that the above assumption can be confirmed. Recent studies [1] indicate that all C. burnetii plasmids encode three proteins that migrate through the secretory system T4SS of C. burnetii, into the phagosome of the host cell, into which C. burnetii actively replicates (hence the survival strategy or virulence of C. burnetii).Recently it was also found that several species of ticks carry bacteria that have been classified to the genus Coxiella by genome sequencing. These species are considered to be obligatory endosymbionts and are classified as Coxiella-like bacteria. Their genome has suffered greater evolutionary reduction than C. burnetii. The probabilities of causing infection in animals and/or humans have not been studied [2].
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Coxiella burnetii synthesizes only one virulence factor (or survival factor), namely the lipopolysaccharide (LPS).
Antigenic variation of LPS is regulated by the C. burnetii genome and expresses forms (phases) of the molecule, phase I and phase II. Phase I bacteria are more virulent while phase II are considered avirulent. Phase I contains sugars not found in phase II LPS. Phase II LPS is the truncated form of LPS I, the missing portion, a product of the deletion of a series of genes involved in the biosynthesis of LPS. Phase I bacteria are encountered in humans, animals and ticks. Phase II bacteria are formed after consecutive passages in cell lines and embryonated eggs (in the laboratory). LPS I affects the early stages of phagocytosis. On the one hand it activates the integrin ανβ3 [leucocytic integrin response (LIC) receptors] on the surface of monocytes and macrophages of the host, and on the other hand it induces the penetration of low numbers of bacteria in the formed phagosome. Simultaneously it disables ανβ2 integrins (TR3 receptors), which induce the penetration of a large number of phase II bacteria in the phagosome. During this procedure (attachment and entry), not only LPS I is involved but two other proteins (the IAP and TLR4 receptors) as well. This protein complex activates the early stage of the immune response, with the production of interferon (IFN)-γ (which induces the Th1 cellular response in the acute form of Q fever). Apart from these proteins, C. burnetii has additional mechanisms that aid the bacterium in surviving and escaping host defense mechanisms. Such a mechanism is inhibition of apoptosis. The inhibition of apoptosis can last up to 4 weeks [3]. Apart from this, it induces a common gene transcription program expressing cytokines, chemokines and other mediators involved in the immune response of the host. This program is reprogrammed depending on the genotype of the strain, the possibilities of survival and any extended or excessive expression of immune response.
Coxiella burnetii produces endospores in order to survive in the environment and spread aerially.
The intracellular cycle of C. burnetii within the phagosome contains two morphologically and functionally distinct forms: the small cell variant (SCV) and the large cell variant (LCV). The metabolism of SCV is characterized as ‘dormant’: its structure resembles bacterial spores. The SCV’s periplasmic space is filled with a dense material, which is considered responsible for the observed resistance of SCV to adverse conditions. The LCV form is metabolically active and is formed under conditions of abundant resources. In acidic pH the SCV is transformed to LCV and when deprivation conditions commence (during the stationary phase of its growth within the phagosome) it is transformed to SCV prior to its release to the environment. Analysis of the proteome of persistently infecting C. burnetii cells indicates that a protein, namely the starvation-sensing protein, is over-expressed in these cells compared with short-term infected cells [4]. We assume that this protein induces the formation of SCV and the survival of C. burnetii in phagocytes and persistent cells, the proteome of which also shows over-expression of other proteins [4]. In collaboration with the group of Heinzen [1], it has been shown further that some of the proteins are expressed by the plasmids of C. burnetii (see MYTH 2); a speculation is that these are involved in bacterial survival through another possible pathway and not the one that leads to the formation of SCV.
Chronic Q fever (endocarditis) is diagnosed exclusively based on Duke’s criteria.
Chronic Q fever diagnosis in patients with a suspicion of endocarditis is a complex search for the presence of predisposing factors (valvulopathies, aneurysms and vasculopathies) and acute Q fever history (history of acute Q fever not only in the last 6 months but also several years ago). Its etiological diagnosis is ensured only by detecting the pathogen using methods such as culture, polymerase chain reaction (PCR) and serological testing. The culture and PCR for C. burnetii are usually negative. Consequently, serological tests remain the key for its etiological diagnosis. Duke’s diagnostic criteria, in their revised versions, set the presence of a phase I IgG titer >1/800 as a major criterion for diagnosis. Professor Raoult’s team [5] suggests the presence of a phase I IgG titer ≥1/6,400 as a major criterion and the presence of a titer between 1/800 and 1/6,400 as minor criterion. An IgG phase I antibody titer of ≤1/800 is indicative of acute disease and represents a typical gene transcription program of a Th2 cell response and M1 macrophages [tumor necrosis factor (TNF), interleukin (IL)-6, IL-12, CCL2, CCL5 and CXC18]. Values ≥1/6,400 indicate chronic Q fever and represent a typical gene transcription program of the Th2 cell response and M2 macrophages [IL-10, transforming growth factor (TGF)-8, IL/rg, MR, CCL16 and CCL13]. Phase I IgG titers between 1/800 and 1/6,400 represent a combination of M1 and M2 responses in an atypical reprogramming of combinatorial Th1 and Th2 cell response transcriptions. The transition from the acute to the chronic form of disease, according to Mege’s team [6], is represented in four stages: M2a, M2b, M2c and M2d. Mege’s proposal is attractive but not clinically documented. However, this zone probably represents the zone of serological uncertainty of 1/800-1/6,400. Using these criteria, I believe that Mege’s M2d stage is the only one that could be characterized as typical chronic Q fever. The zone of serological uncertainty could disappear after the creation of biological markers that represent the clinical forms of Mege’s transition stages. Using Duke’s criteria and criteria created by Holland’s teams, there is a danger of characterizing endocarditis cases not caused by C. burnetii infection as chronic Q fever, as happened in the Holland epidemic.
References
1. Voth D, Beare P, Howe D, et al. The Coxiella burnetii cryptic plasmid is enriched in genes encoding type IV secretion system substrates. J Bacteriol 2011;193:1493-1503.
2. Zhong J. Coxiella-like endosymbionts. Adv Exp Med Biol 2012;984:365-379.
3. Chmielewski T, Tylewska-Wierzbanowska S. Q fever at the turn of the century. Polish J Microb 2012;17:81-93.
4. Vranakis I, De Bock PJ, Papadioti A, et al. Unraveling persistent host cell infection with Coxiella burnetii by quantitative proteomics. J Proteome Res 2011;10:4241-4251.
5. Raoult D. Chronic Q fever: expert opinion versus literature analysis and consensus. J Infect 2012;65:102-108.
6. Benoit M, Benoit D, Mege JL. Microphage polarization in bacterial infections. J Immunol 2008;181:3733-3739.
John Tselendis, Professor, Medical School, University of Crete
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Outbreak news, March 2013
Novel coronavirus infection [1,2,3]The first cases of a novel coronavirus were identified in September 2012. The most recent case, a resident of United Arab Emirates who was transferred for specialist care in Germany, died on 26 March 2013. The global total of confirmed cases is 17, including 11 deaths. Cases have occurred in Jordan, Saudi Arabia and Qatar, and imported cases have been reported in Germany and the United Kingdom. The World Health Organization (WHO) does not advise special screening at points of entry with regard to this event, nor does it recommend that any travel or trade restrictions be applied.
References
World Health Organization (WHO). Available at http://www.who.int/csr/don [accessed 1 April 2013]
European Center for Disease Prevention and Control. Severe Respiratory Disease Associated with a Novel Coronavirus: Rapid Risk Assessment. Available at http://www.ecdc. europa.eu/en/press/news/Lists/News/ [accessed 1 April 2013]
National Travel Health Network and Center (NaTHNaC). Available at http://www.nathnac.org/pro/clinical_updates/coronavirus_270313.htm [accessed 1 April 2013]
Travel Medicine OfficeDepartment for Interventions in Health Care Facilities
Two years of the e-bulletin!
The Hellenic Center for Disease Control and Prevention (HCDCP)’s e-bulletin has already been online for 2 years! It has nearly 10,000 recipients and 20,000 readers via its webpage. So far the e-bulletin has published 453 articles and we would like to thank each of the invited authors individually for honoring us by participating with their valuable contributions. In addition, we would like to thank sincerely our colleagues at HCDCP and CPHL/RPHL. Last but not least we have not forgotten our readers: we thank them for their contributions via the evaluation forms and positive feedback. We are taking into account the comments you have sent us. In case we have inadvertently ‘forgotten’ an author, we apologize in advance and we promise to continue our work with consistency and professionalism in order to help inform health professionals as well as the public regarding public health issues.
G. PetrikkosM. Drogari-ApiranthitouD. KofteridisG. SamonisG. VrioniA. KatragkouE. RoilidesA. VatopoulosN. SpanakisA. TsakrisA. VantarakisE. IosifidisD. KassimosCh. TsigaglouS. Levidiotou-StefanouH. GiamarellouV. PapaevangelouA. KatelarisTh. KaditisS. DoudounakisS. GavriliG. PanosA. P. VasilogiannakopoulosCh. NikolaidisF. Skopouli A. S. AngelidisH. MalisiovaA. G. MavromatisI. S. ArvanitoyannisA. TrichopoulouI. Karydis M. Kogevinas K. KatsouyanniP. Nicolopoulou-StamatiA. AnalitisM. LeotsinidisV. KaraouliP. Theodoraros
G. RachiotisK. SyrrosB. MouchtouriF. TsalikoglouE. KostaraM. Hatzivasileiou PappaE. TzelepiE. PapadogeorgakiG. SpiteriCh. AntoniouD. IoannidisJ. SpiliopoulouG. SaroglouP. ChatzipantaziJ. KosmidesN. VakalisA. MarkaA. D. DiamantidisN. T. PapadopoulosS. AvramidisM. Violaki-ParaskevaA. PsaroulakiA. SeimenisM. TzaniA. KatsiolisG. PappasA. MinasJ. TselentisE. ManesisG. PapatheodoridisE. ArgyropoulosI. ElefsiniotisTh. G. VasileiadisS. HadziyannisM. Raptopoulou-GigiM. Theodoridou G. TzanakakiK. KesanopoulosA. XirogianniV. Papaevangelou
M. Papagrigoriou-TheodoridouM. TsoliaP. TserkezouD. ZoumpoulakisJ. KavaliotisM. PerrakiP. A. KosmidisA. SyggelakisP. Arapandoni-DadiotiV. BarbounisM. Demonakou-VatopoulouP. PapakostasK. PavlakiG. VorgiasCh. N. ForoulisE. VrettouD. KarakasisD. TrichopoulosA. TsakrisSp. A. PournarasG. L. DaikosP. GiakkoupiH. GiamarellouM. K. LazanasP. Gargalianos-KakolyrisN. P. PaissiosV. PapastamopoulosA. SkoutelisE. VogiatzakisS. KarabelaP. IoannidisΚ. KostandinouS. KostandopoulosM. ToumbisS. AnevlavisS. TsiodrasV. PogkaA. KossivakisA. Kalliaropoulos
A. MoutousiM. EmmanouilA. MendisG. SaroglouV. Papaevangelou O. Pappa P. Papastergiou A. BlougouraE. SmetiA. MavridouA. DimopoulosN. VakalisG. ValiakosA. TouloudiCh. IacovakisL. AthanasiouP. BirtsasV. SpyrouCh. BillinisA. PapaS. VellakoulisE. GidarakosA. GkikasM. GkikaT. Gouma-PapadakiD. DiamantidisL. NikolakopoulouP. PetrikosK. PolitiA. SkiadaA. StefosN. B. SypsasD. Chatzigeorgious
Maria Fotinea
Quiz of the month
http://www.keelpno.gr
44
Quiz of the month, March 2013
Why was Q fever named as such?
Send your answer to the following e-mail: [email protected]
The answer to February’s quiz was: Candidiasis.
For more information see: Kurosawa M, Yonezumi M, Hashino S, et al. Epidemiology and treatment outcome of invasive fungal infections in patients with hematological malignancies. Int J Hematol 2012;96:748-757. doi: 10.1007/s12185-012-1210-y. Epub 2012 Oct 31. PubMed PMID: 23111539.
Twenty-three people answered correctly.
Chief Editor:Ch. Hadjichristodoulou
Associate Editors:P. Koukouritakis
Μ. Fotinea
Scientific Board:Ν. Vakalis
Ε. VogiatzakisP. Gargalianos- Kakoliris
Μ. Daimonakou- VatopoulouΙ. LekakisC. Lionis
Α. PantazopoulouV. Papaevagelou
G. SaroglouΑ. Tsakris
Editorial Board:R. Vorou
E. KaratampaniP. Koukouritakis
Κ. MellouD. PapaventsisΤ. PatoucheasV. Roumelioti
V. SmetiCh. TsiaraΜ. Fotinea
Ε. Hadjipashali
Editors:Τ. Kourea- Kremastinou
HCDCP President
T. PapadimitriouHCDCP Director
Graphic Design:Ε. Lazana
Copy Editor:P. Koukouritakis
http://www.keelpno.gr [email protected]
MINISTRY OF HEALTH
HCDCP
HELLENIC CENTER FORDISEASE CONTROL & PREVENTION
MINISTRY OF HEALTH
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