Emerging infectious diseases

SARS-CoV-2

2019
Vectors, climate change and emerging infectious

Climate change

Demographic
Globalization
increase
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Demographic
increase
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Human activity
Pollution
New pathogens
Health system
Climate New infections
change

Ecosistems War
Urbanization modifications

Refugees
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population
Travel
Antibiotic Risk Behaviour
resistance
Flaviviridae, genus flavivirus

▪ > 68 types of viruses

▪ Envelope proteins: E and M

▪ Capsid: icosahedral

▪ Genome: single-stranded RNA (positive polarity)

▪ They are transmitted by arthropods → Arthropod-borne viruses

Diseases spread by animals or with an animal reservoir are called

ZOONOSES
• These viruses have a very broad host range, including vertebrates (e.g.,

mammals, birds, amphibians, reptiles) and invertebrates (e.g., mosquitoes,

ticks).
PHYLOGENETIC TREE OF FLAVIVIRUS
based on complete genome sequence analysis

Ashraf et al., Viruses 2015

The Flavivirus Life Cycle

© MPI Magdeburg (BPE)

 Pathogenesis

• Most flaviviruses infect humans via an insect

(mosquito or tick) bite in the skin. (a)
• Infection is presumed to begin in subdermal
Langerhans cells. (b)
• Infected DC migrate to draining lymph nodes
but also locally produce type I and type II
interferons that limit contiguous spread.
• (c) In the lymph node, flavivirus replication
occurs in a cell type that has not been
definitively identified. Candidate cell types
include macrophages, B cells, and follicular
DC.

http://www.nature.com/icb/journal/v81/n3/fig_tab/icb200330f1.html
 Pathogenesis
• Infectious virus exits the efferent limb and
enters the circulation via the thoracic duct.
• Natural antibodies (IgM), complement, and
possibly NK cells control the initial levels of
virus in the blood.
• Viremia allows spread to secondary visceral
organs (e.g. liver, kidney, spleen) and for
encephalitic viruses, facilitates blood–brain
barrier crossing by an as yet uncharacterized
mechanism.
• Newly induced neutralizing and complement-
fixing IgM and IgG control the extent of tissue
dissemination.
• Infected cells are targeted by the cellular
immune system after recognition by CTL (d).
http://www.nature.com/icb/journal/v81/n3/fig_tab/icb200330f1.html
 The Immune response to flavivirus
Source of antigen

Mature and immature virions induce antibody responses to the Envelope protein, and
these antibodies can function in
➢ neutralization or in
➢ antibody-dependent enhancement of infection
• Antibodies specific for NS1 can interact with membrane-bound NS1 and cause
complement-dependent lysis of virus-infected cells

Rothman et al., Nature Reviews Immunology 2011

 The Immune response to flavivirus
Source of antigen
Newly synthesized viral proteins enter the
MHC class I and II presentation pathways
and viral peptide epitopes are presented on
the cell surface within the binding groove of
MHC molecules

MHC class II molecules present peptides to CD4+ T cells, which principally

produce cytokines but are also capable of lysing infected cells. MHC class I
molecules present peptides to CD8+ T cells, produce cytokines and
principally lyse infected cells
Rothman et al., Nature Reviews Immunology 2011
ZIKV was first isolated from a rhesus monkey in 1947 and from mosquitoes in 1948 in Africa. Afterwards, the
virus was identified within humans in Uganda in 1952.

Envelope
➢ Family: Flaviviridae protein

➢ Genus: Flavivirus Membrane Capsid

protein protein
➢ Virion: enveloped virus with an icosahedral
Genomic
shell RNA

➢ Genome: single-stranded RNA of about

Single stranded
11,000 bases (positive polarity) RNA

Polyprotein precursor
The genome encoding a polyprotein that is
processed into three structural proteins the
C prM E NS1 NS2A NS2B NS3 NS4A NS4B NS5
capsid (C), the precursor of membrane (prM),
and the envelope (E), and seven nonstructural
proteins.
Although the isolation of ZIKV has so far been confined to the African continent, serological
evidence has shown widespread distribution of the virus even in Asian countries such as
Malaysia, India, Philippines, Thailand, Vietnam, Indonesia, and Pakistan (Li MI et al., PLoS 2012)

Global map of the areas with current or past evidence of Zika Virus
(Centers for Disease Control and Prevention)
Many different Aedes species mosquitoes
can account for the transmission of ZIKV:

➢ The earliest evidence of ZIKV in a pool of Ae.

africanus from Uganda in 1948 coincides with

its first isolation from a rhesus monkey in the

same location.

➢ Early experimental studies demonstrated the

competency of Ae. aegypti to transmit ZIKV

(Boorman et al. and Cornet et al.)

 Nonvector arbovirus transmission

➢ directly between vertebrates

➢ sexually

➢ from mother to child

➢ by hospitalization

➢ by transfusion

➢ via bone marrow

➢ via organ transplantation

Mother to child
transmission :

➢ A mother already infected with Zika virus near the time of delivery can pass on

the virus to her newborn around the time of birth → this is rare.

➢ In 2015, Zika virus RNA was detected in the amniotic fluid of two fetuses,

indicating that it had crossed the placenta and could cause a mother-to-child

infection → Zika virus could pass from mother to fetus during pregnancy.

➢ No reports of infants getting Zika virus through breastfeeding.

 Sexual Transmission
There have been only three documented cases linking Zika to a sexual

intercourse:

❖ In 2008, Colorado → microbiologist named Brian Foy contracted Zika after

travel to Senegal; his wife came down with the disease a few days later even

though she had not left northern Colorado and was not exposed to any

mosquitoes carrying the virus.

❖ In 2013, French Polynesia → semen and urine samples from a 44-year-old

Tahitian man tested positive for Zika even when blood samples did not.

❖ In 2016, Texas → a patient who had a sexual intercourse with someone who

had recently returned from Venezuela infected with the mosquito-borne virus.
 Tang H. et al., 2016

The direct target cells of ZIKV in the

developing human fetus are not clear.
This study shows that:
❑ A strain of the ZIKV, MR766, serially
passaged in monkey and mosquito cells
efficiently infects human neural progenitor
cells (hNPCs) derived from induced
pluripotent stem cells.
 Clinical symptoms

➢ Low-grade fever (between 37.8C and 38.5C )

➢ Rash

➢ Non-purulent conjunctivitis

➢ Arthritis/arthralgiamyalgia

➢ Mild headaches and retro-ocular Headaches

➢ Fatigue/lethargy/asthenia

➢ Strawberry tongue and red, cracked lips

➢ Swollen lymph node in neck

➢ Red palms/soles and swollen hands/feet

 Complications

ZIKV infection was associated with only mild illness prior to the large French Polynesian outbreak in
2013 and 2014, when severe neurological complications were reported, and the emergence in Brazil of a
dramatic increase in severe congenital malformations (microcephaly) suspected to be associated with
ZIKV.

Neurological complications in adults:

▪ Guillain-Barré syndrome (GBS), inflammation of multiple nerves, characterized by temporary muscle
weakness, loss of sensata and possibly paralysis.

Neurological complications in neonates:

▪ Meningoencephalitis, inflammation of the brain and the membranes covering it.

Autoimmune complications:
▪ Thrombocytopenic purpura, purplish skin discoloration that is characterized by bleeding into the
and by hemorrhages into mucous membranes and associated with a reduction in circulating blood
platelets and prolonged bleeding time.
▪ Leukopenia, reduction in the number of circulation white blood cells in the blood.
 Diagnosis

➢ The clinical presentation of Zika fever is not specific and can mimic diseases

responsible for fever, rash, and arthralgia, especially dengue and chikungunya.

→ highlighting the importance of laboratory investigation of patients presenting

with “dengue-like syndromes” and testing negative for dengue.

➢ RT-PCR testing be done on serum collected within 1 to 3 days of symptom onset

or on saliva or urine samples collected during the first 3 to 5 days.

➢ Later on detection of specific IgM and IgG antibodies.

 Treatment

❑ There is NO VACCINE that can treat Zika infections and prevent it;

You can treat symptoms by doing these common things:

❑ Acetaminophen and paracetamol to relieve fever;
❑ Antihistaminic to control pruritic rash;
❑ Patients should be advised to drink plenty of fluids to replenish fluid lost from sweating,
vomiting and other insensible losses;
❑ Treatment with acetylsalicylic acid and nonsteroidal anti-inflammatory drugs is
discouraged to the reported increased risk of hemorrhagic syndrome with other
flaviviruses;
❑ Do not take non-steroidal anti-inflammatory drugs and aspirin
In the first days after symptom onset (viremic phase),
patient isolation, to avoid further mosquito bites, is
recommended to prevent the transmission to other
persons
 Prevention tips

Prevention and control relies on reducing mosquitoes through source

reduction and reducing contact between mosquitoes and people.

❖ Special attention and help should be given to those who may not be able to

protect themselves adequately, such as young children, the sick or elderly.

❖ During outbreaks, health authorities may advise that spraying of insecticides

be carried out.

❖ Travellers should take the basic precautions described above to protect

themselves from mosquito bites.

 ➢ Family: Flaviviridae
➢ Genus: Flavivirus
➢ Virion: Enveloped virus with icosahedral capsid
➢ Genome: single-stranded RNA with positive polarity,
10,760-11,008 nucleotides in length

The genome has a single large open reading frame that encodes 10 proteins:
▪ 3 structural proteins encoded at the 5’ of viral genome
▪ 7 nonstructural (NS) proteins encoded by the remaining coding region
Several different species of mosquitoes transmit the virus

Aedes aegypti mosquitoes Haemogogus mosquitoes

➢ Non-human primates can be a reservoir

➢ Vertical transmission
 Transmission

There are 3 types of transmission cycles:

1. Sylvatic (or jungle) yellow fever involving

mosquitoes transmitting virus through
monkey intermediates to other mosquitoes
with the occasional human infection;
2. Intermediate (or Savannah) yellow fever
involve humans and monkeys surviving as
intermediates with minor outbreaks
occurring;
3. Urban cycles yellow fever, the largest of
epidemics, where infected humans introduce
the virus to new mosquito population and
non-immune people.
❑ There are an estimated 84,000–170,000 cases and up to 60,000 deaths due to yellow fever
per year.
❑ The virus is endemic in tropical areas of Africa and South America, with a combined
population of over 900 million people.
 Why has yellow fever never been seen in Asia or
Pacific regions?

• The mosquito vector Ae. Aegypti is prevalent in Asia and Pacific countries.

• Laboratory studies indicate that Asian strains of Ae. Aegypti can transmit

YFV but are less competent than strains from the Americas.

Demographic factors, including the remote location of sylvatic YF transmission

and the cross-protective immunity provided by prior exposure to dengue and

other flaviviruses, likely play a role in the lack of YF in Asia.

 Clinical Symptoms
➢ Fever

➢ Chills

➢ Flu-like symptoms (muscle aches, severe headache)

➢ Back pain

➢ Nausea

➢ Weaness

➢ Jaundice (from the damaged liver)

➢ Hepatomegaly

➢ Bleeding from the eyes, nostrils, anus and other mucous membranes.

➢ Black-colored and blood-filled vomit

➢ Deterioration of the liver, kidneys, and heart.

 Clinical features

▪ The incubation period after mosquito bite is usually 3–6 days

▪ Yellow fever is an acute disease with brief duration and three different periods:

1. Period of infection

2. Period of remission (starts 3-4 days after onset of the first symptoms)

3. Period of intoxication (on the 3th to 6th day after the onset of infection,

20-30% of the patients who enter this period die)

Beasley D.W.C. et al.; Antiviral research, 2015

 Liver involvement
➢ The liver is the main target organ, but other tissues (especially kidneys) become

infected, Kupfeer cells are initially infected

➢ Hepatocytes are infected directly via Kupffer cells or haematogenously

➢ Eosinophilic degeneration with Councilman bodies (apoptosis), rather than by ballooning

and necrosis seen in A-E hepatitis

➢ In fatal cases, amorphous masses (Torres bodies) or small, granular, yellow bodies

(Villela bodies) can also be seen. Not diagnostic.

➢ Liver cells death is due to apoptosis (Councilman bodies)

- Hepatocyte in the midzone of the lobe express Fas ligand and lymphocytes

infiltrated the liver mediate apoptosis

Quaresma. Virology 2006

➢ There is no disruption of the reticular architecture of the liver, even in fatal cases.
 Liver involvement

➢ AST levels > ALT, probably due to viral injury to the myocardium and skeletal

muscle (useful to distinguish from other viral hepatitis).

➢ Levels are proportional to disease severity and might remain elevated for up to

2 months after onset.

Oudart. Bull World Health Organ 1979

➢ Advanced disease → encephalopathy

➢ Global reductions in clotting factors synthesized by the liver → contribute to

bleeding.
 Diagnosis
➢ Yellow fever is difficult to diagnose, especially during the early stages.

➢ It can be confused with severe malaria, dengue hemorrhagic fever, leptospirosis, viral

hepatitis (especially the fulminating forms of hepatitis B and D), other hemorrhagic fevers

(Bolivian, Argentine and Venezuelan hemorrhagic fevers as well as other Flaviviridae such as

the West Nile and Zika viruses) and other diseases, as well as poisoning.

➢ Blood tests can detect yellow fever antibodies produced in response to the infection.

➢ Several other techniques are used to identify the virus in blood specimens or liver tissue

collected after death. These tests require highly trained laboratory staff and specialized

equipment and materials

 Laboratory Diagnosis

Virus isolation
• from the blood or post-mortem liver tissue

Rapid diagnostic test:

• Detection viral genome by PCR in blood o tissues

Detection IgM antibodies (ELISA)

• IgM for acute phase, coupled with convalescent antibodies (IgM/IgG)
• Neutralization Ab are more specific for YF
Prevention

❖ immunization

❖ avoid mosquito bites

❖ wear long-sleeved shirts & long pants

❖ use insect repellant

❖ spray living areas with insecticide

❖ use bed net when sleeping in infested

area
 Treatment

There are no approved therapies

❖ Drink plenty of fluids

❖ Maintain nutrition and prevent hypoglycemia

❖ Supplemental oxygen

❖ Dialysis

❖ Therapeutic products under investigation fall into three major categories:

1. polyclonal or monoclonal antibody preparations that typically target the viral E protein;

2. compounds targeting viral proteins involved in replication;

3. immunomodulatory compounds that boost host antiviral responses.

 Vaccine of Yellow Fever

➢ Virus first isolated in 1928

➢ YFV 17D live attenuated vaccine strain derived 1937

➢ 31 amino acid differences between vaccine strain and wild-type

➢ Used for the prevention of yellow fever in travelers, for routine immunization of infants in

endemic areas, for catch-up campaigns in Africa, and for emergency response during

outbreaks.

➢ Protective immunity occurs in 90% within 10 days and in nearly 100% within 3-4 weeks.

➢ Immunity is lifelong after a single dose.

➢ Adverse events : 43/100,000

Department of Health and Human Services Centers for Disease Control and Prevention 2010
 Protection of travellers visiting areas with risk
of Yellow Fever

The risk for travelers is determined by multiple factors: vaccination status,

location of travel, duration exposure…

Vaccine should be offered:

➢ to all unvaccinated travellers aged ≥ 9 months, travelling to and from at-risk
areas, unless they belong to the group of individuals for whom YF vaccination
is contraindicated.

➢ to travellers going to areas with low risk of YF transmission, although the

vaccination is generally not recommended, it might be considered for the ones
travelling for a long period to areas where they may be exposed to heavy load
of mosquito bites.
 Yellow Fever Initiative

➢ The number of yellow fever cases has been decreasing over the past 10 years since the
launch of Yellow Fever Initiative in 2006.

➢ The Yellow Fever Initiative, led by WHO and UNICEF with the support of the Global
Alliance for Vaccines and Immunization (The GAVI Alliance), has now been launched with
the aim of dramatically reducing the risk of yellow fever outbreaks in 12 endemic
countries in Africa through the vaccination of 48 million people over the coming four
years.
✓ Family: Flaviviridae
✓ Genus: Flavivirus
✓ Genome: single stranded RNA virus with positive
polarity
10 genes
3 structural 7 non-structural
(C,M and E) (NS1, NS2A, NS2B, NS3, NS4A,
NS4B, NS5)

✓ There are four antigenically different serotypes (DENV1-4) of the virus;

there is report that a fifth serotype has been found (Mustafa M.S., MJAFI, 2015)
 Global burden of Dengue virus
• The actual numbers of dengue cases are underreported and many
cases are misclassified.
• Dengue has seen a 30-fold upsurge worldwide between 1960 and 2010, due
to:
• increased population growth rate,
• global warming,
• unplanned urbanization,
• inefficient mosquito control,
• frequent air travel,
• lack of health care facilities.
(Hasan et al., J Int Soc Prev Comm Dent. 2016)

• One recent study estimates 390 million dengue infections per year, of
which 96 million with clinical manifestations. (Bhatt et al., Nature 2013)
 Dengue endemic regions

• Dengue virus is found in tropical and sub-tropical climates worldwide, mostly in

urban and semi-urban areas.
WHO, World Health Organization 2016
In Italy

ISS, Istituto Superiore di Sanità, 2015

 Etiopathogenesis

▪ Dengue virus is transmitted by female

mosquitoes mainly of the species Aedes
aegypti and, to a lesser extent, Ae. albopictus.

▪ Humoral, cellular, and innate host immune

responses are implicated in the progression
of the illness and the most severe clinical
signs occur following the rapid clearance
of the virus from the host organism.

Hasan et al., J Int Soc Prev Comm Dent. 2016

 Etiopathogenesis

▪ Alterations in endothelial microvascular

permeability and thromboregulatory
mechanisms lead to an increased loss
of protein and plasma.
▪ Proposed theories suggest that
endothelial cell activation caused by
monocytes, T-cells, complement
system, and various inflammatory
molecules mediate plasma leakage.

Hasan et al., J Int Soc Prev Comm Dent. 2016

 Classification of Dengue fever (DF)

• According to the 1997 classification, dengue fever can be divided into:

❖ Undifferentiated fever (DF)
❖ Dengue haemorrhagic fever (DHF)
DHF was further subdivided into grades I–IV:
Grade I: Only mild bruising or a positive tourniquet test
Grade II: Spontaneous bleeding into the skin and elsewhere
Grade III: Clinical sign of shock
Grade IV: Severe shock - feeble pulse, and blood pressure cannot be
recorded.
Grades III and IV comprise Dengue shock syndrome (DSS)

Hasan et al., J Int Soc Prev Comm Dent. 2016

 Treatment of DENV infection

There is no specific treatment for dengue fever

However…

Fluid replacement and antipyretic therapy with paracetamol is the

preferred therapy following the febrile phase

Hasan et al., J Int Soc Prev Comm Dent. 2016

 Immunization

o In late 2015 and early 2016, the first live attenuated tetravalent dengue

vaccine, Dengvaxia (CYD-TDV) by Sanofi Pasteur, was registered in several

countries for use in individuals, living in endemic areas.

o 3 other vaccine candidates (based on subunit, DNA, and purified inactivated

virus platforms) are at earlier stages of clinical development.

WHO, World Health Organization 2016

 Immunization

o CYD-TDV is a live attenuated tetravalent chimeric vaccine made using recombinant DNA

technology by replacing the PrM (pre-membrane) and E (envelope) structural genes of

the yellow fever attenuated 17D strain vaccine with those from each of the four dengue

serotypes. Ongoing phase III trials in Latin America and Asia involve over 31,000 children

between the ages of 2 and 14 years. In the first reports from the trials, vaccine efficacy

was 56.5% in the Asian study and 64.7% in the Latin American study in patients who

received at least one injection of the vaccine. Efficacy varied by serotype.

 ✓ Family: Flaviviridae
✓ Genus: Flavivirus
✓ Icosahedral nucleocapsid which is contained
in a lipid bi-layered envelope (50 nm)
✓ Genome: single-stranded RNA virus with
positive polarity
✓ Viral nonstructural proteins: NS1, NS3, NS4A, NS4B, NS5, are responsible for
regulating viral mechanisms of transcription, translation and replication
and attenuate host antiviral responses
 Outbreaks of WNV

❖ First isolated in a woman in the West Nile district of Uganda in 1937

❖ It was identified in birds in Nile delta region in 1953.
❖ Endemic in Africa and Middle East Europe:
- France (1962-65), Eastern Europe (1996-1999), Israel (2000)
❖ In 1999 a WNV circulating in Israel and Tunisia was imported in
New York producing a large and dramatic outbreak that spread
throughout the continental United States of America (USA) in the
following years.

WHO, World Health Organization 2016

 Outbreaks of WNV in the U.S.A
The WNV outbreak in USA (1999-2010) highlighted that:
importation and establishment of vector-borne pathogens outside their current
habitat represent a serious danger to the world.

2002 2003

2004 2005

CDC, Department of health & human services-USA website 2016

In Italy

ISS, Istituto Superiore di Sanità, 2015

 Transmission

1. Human infection is most often the result

of bites from infected mosquitoes.
2. Mosquitoes become infected when
they feed on infected birds, which
circulate the virus in their blood for a
few days.
3. The virus eventually gets into the
mosquito's salivary glands.
4. During later blood meals (when mosquitoes bite), the virus may be injected into
humans and animals, where it can multiply and possibly cause illness.

N.B. Horses, just like humans, are “dead-end” hosts, meaning that while
they become infected, they do not spread the infection
WHO, World Health Organization 2016
 Transmission

• The virus may also be transmitted through contact with other infected

animals, their blood, or other tissues.

• A very small proportion of human infections have occurred through organ

transplant, blood transfusions and breast milk.

• There is one reported case of trans-placental (mother-to-child) WNV

transmission.

• To date, no human-to-human transmission of WNV through casual

contact has been documented.

WHO, World Health Organization 2016

 Pathogenesis

➢ Infection with WNV is either asymptomatic (no symptoms) in around

80% of infected people

➢ About 20% of people who become infected
with WNV will develop West Nile fever.

➢ It is estimated that approximately 1 in 150 persons infected with the

WNV will develop a more severe form of disease.

WHO, World Health Organization 2016

Symtoms of West Nile fever

• Mild flu-like illness with

- malaise
- eye pain
- headache
- myalgia
- gastro-intestinal discomfort
- rash

WHO, World Health Organization 2016

 Pathogenesis of severe form of WN disease

Epidemiological studies indicated that the frequency and severity of

clinical illness increases with age

Three distinct stages of WNV pathogenesis have been defined:

1. EARLY PHASE: initial infection and spread

2. VIREMIC PHASE: peripheral virus amplification
3. CENTRAL NERVOUS SYSTEM (CNS) PHASE: neuroinvasion,
causing meningo-encephalitis

Suthar and Pulendran, Curr Opin Virol. 2014;

Lim et al., Viruses 2011
 Pathogenesis of severe form of WN disease
GLIA
2
LIMITANS
ASTROCYTE

NEUTROPHILIS
VIRAL LOAD
MICROGLIA

MONOCYTES

ENTRY OF IMMUNE CELLS

WNV DISSEMINATION

1 NEURON

SPACE
PERIVASCULAR

1. Recent data suggest that leukocytes have to pass two barriers in order to
arrive in the brain parenchyma: the endothelial cell vascular wall (1) and the
glial limitans (2), collectively referred to as Blood Brain barrier
Lim et al., Viruses 2011
 Diagnosis of WN infection

West Nile virus can be diagnosed by a number of different tests:

• IgG antibody sero-conversion in two serial specimen collected at a
one week interval by enzyme-linked immunosorbent assay (ELISA)
• IgM antibody capture enzyme-linked immunosorbent assay (ELISA)
(IgM can be detected in nearly all cerebrospinal fluid (CSF) and serum specimens
received from WNV infected patients at the time of their clinical presentation; serum
IgM antibody may persist for more than a year)

• neutralisation assays
• viral detection by reverse transcription polymerase chain reaction
(RT-PCR) assay
• virus isolation by cell culture
WHO, World Health Organization 2016
 Treatment and vaccine

NO VACCINE IS AVALABLE FOR HUMANS

Treatment is supportive for patients with neuro-invasive West Nile

virus and provides:
• hospitalization
• intravenous fluids
• respiratory support
• prevention of secondary infections

WHO, World Health Organization 2016

 Reducing the risk of infection in people

In the absence of a vaccine,

the only way to reduce infection in people is:
• by raising awareness of the risk factors
and
• educating people about the measures they can take to reduce
exposure to the virus (use of mosquito nets, personal insect
repellent, by wearing light coloured clothing and by avoiding outdoor
activity at peak biting times).

WHO, World Health Organization 2016

Aedes aegypti - current known distribution - January 2018
 Historical distribution of Ae.aegypti
in Europe up to 1960
• Ae. aegypti was present until 1950-60 in all
Mediterranean Countries and in all Black sea
countries, and disapeeared in 1960.
• It was found in Italy for the last time in 1972
Callot J, Delecolle J-C. Ann Parasitol Hum Comp 1972; 47: 665
• Ae.aegypti is to date absent in West Europe, but it
colonized again the black sea costs in 2008
Yunicheva YU,et al.Med Parazitol Parazit Bol 2008; 3: 40–43
 Dengue outbreak in Guangzhou, 2014
• Mainland China had 55,114 reported dengue cases
from 2005 to 2014, of which 47,056 occurred in
2014.
• 94% of the indigenous cases were reported in
Guangdong Province, 83% of which were in Guangzhou
City.
• An early imported case was the most important
factor in determining the 2014 outbreak.
exclamation mark
• Extraordinary high precipitation in May and August,
2014 appears to have increased vector abundance.
Cheng Q, et al. PLoS Negl Trop Dis 2016; 10: e0004417.
 Chikungunya in Italy, 2017
• •AlActive from 2017,
6 ottobre March282to notifiche (156 confermati e
September.
126 probabili) in Lazio (Anzio, Roma e Latina-242
•casi probabili e confermati)
In appropriate conditions ofe Calabria (Guardavalle
Marina-33
temperaturecasiand
probabili e confermati).
humidity, the
adult
• Casi female lives
‘importati’ 30-40 days. (4 casi di malattia
in Emilia-Romagna
•3 In
probabili da Guardavalle
areas with cold wintersMarina
the e uno confermato
adeggs
Anzio),
that Marche
enter in(un caso da Anzio), Francia (1
diapause,
caso
thatconfermato da Anzio)
is, block their e Germania (un caso
maturation
probabile con storia
in the winter monthsdi to
viaggio a Roma).
reactivate then in spring
FILOVIRUSES
Serologic Evidence of Fruit Bat Exposure
to Filoviruses, Singapore, 2011–2016
• Screening of 409 serum samples from bats of 3 species
(Cynopterus brachyotis, Eonycteris spelaea, and
Penthetor lucasi), by using a multiplex assay that detects
antibodies against filoviruses.
• Positive samples reacted with glycoproteins from
Bundibugyo, Ebola, and Sudan viruses, indicating filovirus
circulation among bats in Southeast Asia.

1, Zaire ebolavirus; 2, Bundibugyo ebolavirus; 3, Taï Forest ebolavirus; 4, Sudan

ebolavirus; 5, Reston ebolavirus–monkey; 6, Reston ebolavirus–pig; 7, Marburg virus–
Musoke; 8, Marburg virus–Angola; 9, Ravn virus.
Laing ED et al. EID 2018; 24: 122-126
Ebolavirus
Ebola Virus Disease (EVD) or Ebola Hemorrhagic Fever (EHF)
 Viral Characteristics

-Filamentous, circular, coiled, ‘U’ or ‘6’- like virions of ≈1,028 nm in length.

- The virus has an envelope containing the virally encoded GP glycoprotein for target
cell recognition.
-Helicoidal nucleocapsids : 900-1200 nm in length, and 45-100 nm in diameter, with
a central canal of 20–30 nm in diameter, composed by >3000 copies of the
nucleoprotein
(Adapted from Jin Huk Choi BioDrugs. 2014)
 Genome
• Negative-stranded RNA linear genome of 18-19 Kb

VP35: polymerase complex protein→involved in viral genes expression. Interacts with

NP protein
VP40: Matrix protein→ for assembly and budding
VP24: Second structural matrix protein→ often associated with viral envelope
N/NP: Nucleoprotein (739 nm, the largest nucleoprotein)
VP30: Minor nucleocapsid protein
GP: Surface glycoprotein → binding to cellular receptors
L: RNA dependant RNA polymerase
(Viralzone 2015, Swiss Institute of Bioinformatics)
 Replicative cycle
In 8 hours→it spreads rapidly

ViralZone Sib Swiss of Bioinformatic 2014

 Replication and transcription model of filovirus: EBOV vs
Marburg virus (MARV)
Cytoplasmatic replication and transcription
(1) Transcription initiation: polymerase (L
and VP35 ) binds to a promoter site in the
leader region and transcripts NP . The
first 23 NTs of the nascent mRNA form a
stable secondary structure kept by VP30
(1) (2) Transcription antitermination due to
(2) the RNA secondary structure is abrogated
by VP30 (t may be speculated that VP30
resolves the RNA structure)
(3)
(3) Elongation and transcription
reinitiation of other genes is independent
of VP30.

• Transcription of viral genes results in the synthesis of 7 monocistronic capped and

polyadenylated mRNAs.
• For MARV transcription, NP, VP35 and L are sufficient, while EBOV transcription is
dependent on the transcription activator VP30. Adapetd from Elke Mühlberger Future Virol. 2007
 Taxonomy
Order Family Genus Species Virus Abbreviation Discovered
Bundibugyo ebolavirus Bundibugyo Virus BDBV Uganda, 2007
Reston ebolavius Bundibugyo Virus RESTV Virginia, USA 1989
Sudan ebolavirus Sudan Virus SUDV Sudan, 1976
Mononegavirales Filoviridae Ebolavirus
Tai Forest ebolavirus Tai Forest virus TAFV Ivory Coast, 1995
Zaire (Congo), 1976,
Zaire ebolavirus Ebola virus EBOV
Ebola river

-Only Bdbv, Ebov e Sudv have been responsible of important epidemics in Africa
-EBOV: causative agent of the 2014 West Africa epidemic

Criteria inclusion:
• several overlap of genes
• GP encodes 4 proteins (SGP, ssGP, Δ-peptide and GP1,2) using a transcriptional
co-cutting to get ssGP and GP1,2 and proteolytic cleavage to get SGP and Δ-peptide
• the peak of its infectivity is associated with particles of ≈805 nm in length
• its virions do not show almost antigenic cross-resistance with virions marburg
 Criteria inclusion: Ebolavirus phylogenetic tree

http://bioinformatics.cvr.ac.uk/paleovirology
/site/html/non_retroviral_eves.html

Carrol A.S Journal of Virology 2016

5 different strains of Ebola virus are
known:

Bundibugyo ebolavirus (Bdbv)

Zaire ebolavirus (Ebov) → Causative agent of the 2014
Reston ebolavirus (Restv) West Africa epidemic
Sudan ebolavirus (Sudv),
Taї Forest ebolavirus (Tafv)

→Only Bdbv, Ebov e Sudv have been

responsible of important epidemics in
Africa
 Where Does the Ebola Virus Come From?
• Virus-infected bats: the most likely reservoir
• Spillover event from infected wild animals (e.g., fruit bats, monkey, duiker)
to humans, followed by human-human transmission
Ebola Virus Transmission

• Virus present in high quantity in blood, body fluids, and excreta of

symptomatic EVD-infected patients

• Opportunities for human-to-human transmission

-Direct contact (through broken skin or unprotected mucous membranes) with
EVD-infected patient’s blood/body fluids
-Sharp injury
- Direct contact with corpse of person died of EVD
- Indirect contact with EVD-infected patient’s blood/body fluids via
contaminated materials

• Ebola transmission via contact with blood, fluids or meat of infected

animals
- Limited evidence that dogs become infected with Ebola virus
- No reports of dogs or cats becoming sick with Ebola virus
Ebola Virus in Breast Milk in an Ebola Virus–Positive Mother with Twin Babies,
Guinea, 2015

Nordenstedt H, Emerg Infect Dis. 2016

 Human-to-Human Transmission
• Infected persons are not contagious until onset of symptoms
• Infectiousness of body fluids increases as patient becomes more ill
• Transmission via inhalation (aerosols) has not been demonstrated
CBC News Last Updated: August 11, 2014
Early Clinical Presentation
Pathogenesis
 Primary targets of Ebola replication such as antigen presenting cells
(APCs), macrophages and dendritic cells (DCs) become deregulated and
unable to express co-stimulatory molecules or stimulate naïve T cells

Feldmann H, Lancet. 2011

Infection of Monocyte-derived human macrophages and peripheral
blood mononuclear cells explain the rapid transport to other tissues

• Infection of
macrophages/monocytes
stimulates “cytokine storm”
(TNF-α , IL-1β, 2, 6, 8 and 10,
ROS and NO,and MIP-1a);

• It recruits additional APCs to

the site of infection→more
hosts to support virus
replication

• It contributes to the
pathogenesis by increasing
endothelial permeability, and
vascular leakage

→rapid dissemination of infected APCs throughout the systemic circulation;

→release of Ebola in secondary lymphoid organs, lungs, liver and other
ancillary sites (Adapted from Jin Huk Choi BioDrugs. 2014)
Ebolavirus plague over time
 2014 EVD Epidemic
8 affected countries: Guinea, Liberia, Mali, Sierra Leone, Spain and the United States
of America, Nigeria, Senegal
3 Countries with widespread and
intense transmission

•Guinea: 1 731 cases and 1 041

deaths as of 2 November 2014;

•Liberia: 6 525 cases and 2 697

deaths as of 31 October 2014;

•Sierra Leone: 4 759 cases and 1

070 deaths as of 2 November
2014;

WHO Ebola Roadmap, 05 Nov 2014

The spread of the deadly Ebola virus since 1976 in Africa

CBC News Last Updated: August 11, 2014

EVD is crossing the borders

Italy
2015
 Resulting key elements about EVD

• Different courses depending on viral species

• Rapid death
• Rapid personal diffusion
• Transmission through corpse (funeral patient case)
• Monkeys/forest origin: nutrition of dead chimpanzee→ EDV in the past referred more to
poor/indigenous populations
• initial medical malpractice and inadequate laboratory equipment
• From 2012, more care by healthy organizations
• Cases of patients who overcome the illness
• Necessity of travel control worldwide
Ebola Virus Diagnosis
 The high mortality rate and the lack of adequate vaccines and
therapies, classify the genus Ebolavirus as a biohazard agent level 4,
as well as Category A bioterrorism agent

Filoviruses have been

implemented in biological
warfare; Soviet Russia declered
to have had used them in their
biological weapons programme.
No particular attention and therapeutical solutions in serveral years
until now…
Generalized approach to Ebola Vaccine Development: overexpression
of Ebola glycoprotein and/or nucleoprotein
Administration of replication
deficient recombinant
adenoviruses/plasmid vectors or
attenuated recombinant viruses
(VSV or HPIV3) bearing Ebola
glycoprotein.
Transduction of various cellular
targets; increasing Ebola antigens
that enter the general circulation
Glycoprotein and nucleoprotein
produced are processed by
macrophages and dendritic cells

Generation of strong B and T cell-

mediated immune responses
against Ebola

(Adapted from Jin Huk Choi BioDrugs. 2014)

 Ebola Vaccine Platforms Currently Tested In Non-Human Primates

VP: total number of virus particles (infectious and non-infectious); PFU: plaque forming units;
PU: particle units; TCID50: median tissue culture infective dose; FFU: focus forming units

(Adapted from Jin Huk Choi BioDrugs. 2014)

• Merck's vaccine, VSV-ZEBOV, is a vesicular stomatitis attenuate virus designed to
express a glycoprotein of the Zaire strain of Ebola.
• After successful trials in macaques, the trial "Ebola ça Suffit" or "Ebola, that’s
enough” conducted over 7,500 people in in Guinea by the WHO.
• The vaccine has shown 100% efficacy.
RVEBO® (Ebola Zaire Vaccine, Live also known as V920,
rVSVΔG-ZEBOV-GP or rVSV-ZEBOV) is approved by the U.S.
Food and Drug Administration (FDA) for the prevention of
disease caused by Zaire ebolavirus in individuals 18 years of
age and older as a single dose administration. ERVEBO is a
replication-competent, live, attenuated recombinant vesicular
stomatitis virus (rVSV) vaccine manufactured by Merck. It is
not possible to become infected with EBOV from the vaccine
because the vaccine only contains a gene from the Ebola
virus, not the whole virus. Specifically, it contains a gene for
the EBOV glycoprotein that replaces the gene for the native
VSV glycoprotein. ERVEBO does not provide protection
against other species of Ebolavirus or Marburgvirus.
Zhao Z.,et al. BMC Bioinformatics 2016
 Ebola VP24 interacts with human Karyopherin- α to disarm the human
immune system: VP24 inhibition may disrupt the VP24-Karyopherin-α
interaction and reduce the virulence of Ebola.
Structural systems pharmacology
approach

The binding interface of VP24 with Karyopherin-α.

-An open pocket is in yellow spheres.
-Initial conformation from PDB and conformation
generated from MD simulation is in grey and blue,
respectively.
Adapted fromZhao Z.,et al. BMC
-In red is the loop (amino acids 181–186) that has a
Bioinformatics 2016 prominent conformational change after MD simulation.
 Indinavir, an HIV protease inhibitor, may also reduce the virulence of
Ebola based on it high binding affinity to VP24

It was ranked second in the protein-ligand docking study

The predicted binding mode of Indinavir in VP24 of Ebola

• VP24 binding pocket accommodates Indinavir depicting 3 hydrogen bonds between
Indinavir and VP24:
1) the O2 atom of Indinavir and the nitrogen of residue Gln94 in VP24
2) the atom N4 of Indinavir and the oxygen atom in the sidechain of VP24’s Gln94
3) the O4 atom of Indinavir and the oxygen atom from the main chain of Gln94 of
VP24.
• Notably, 11 amino acids form hydrophobic interactions with Indinavir.

Adapted fromZhao Z.,et al. BMC Bioinformatics 2016

 It is possibile to heal by EVD: the Italian cases

Stefano Marongiu, Emergency

Fabrizio Pulvirenti, Emergency medician nurse, was infected by EBOV. He
is the first Italian infected patient, healed was treated with the same
after 39 days of admission at Spallanzani guidelines of Pulvirenti
Hospital

Basic Guidelines:
• Mechanical ventilation and antimalarial therapy appear to be indispensable.
• Experimental treatments consisted of convalescent plasma, favipiravir, monoclonal
antibodies and ZMab melanocortin. However, further research is needed.
• Immediately after administration ZMAb , viremia declined significantly.
World’s deadliest animals?
RANK
 Potential geographic distribution patterns of Ae.
aegypti in 2050 under a moderate emissions scenario.

• Daily
Studies
mean
generally
temperature
projectand
that,as
variation
temperatures
in temperature
continue
aretotwo
rise
ofand
the
most important
precipitation patterns
driverschange,
of the current
opportunities
distribution
are increasing
and incidence
for of
dengue. geographical
further Precipitation expansion
and precipitation
of Aedes extremes,
vectors drought or excess
rainfall,also affect mosquito abundance and arbovirus incidence.
Ebi and Nealon Environmental Research 2016; 151: 115–23
Vector-borne infectious diseases that may
be affected by climate changes in Europe.

Vonesch et al.Ann Ist Super Sanita 2016; 52: 397-405

Aedes koreicus - current known distribution -
January 2018

Found in Belluno during 2011. Recently reported in Lombardy and

Genova,is a possible efficient transmitter of Dirofilaria immitis to
dogs
 TOGAVIRIDAE

Outer shell: phospholipid

The virions are characterized bilayer with glycoproteins
E1 and E2
by :
▪ Pericapsid of a diameter of
60-70 nm, with 2
glycoproteins (E1 and E2) Icosahedral
nucleocapsid: C protein

▪ Icosahedral capsid
composed by protein C
Genome single-strand
RNA positive-sense
▪ Genome single-strand
RNA, sense + (~10-12 kb)
with 5’ cap and 3’ poly-A
G.Antonelli e M.Clementi - Principi di Virologia Medica (2012)
 CLASSIFICATION OF
TOGAVIRIDAE

Togaviridae

Alphavirus Rubella
(27 members, 11
pathogenic to
virus
humans)
 STRUCTURE

ALPHAVIRUS RUBELLA VIRUS

▪ 4 non-structural protein ▪ 2 non-structural protein
▪ 4 structural protein ▪ 3 structural protein

Alphavirus

http://nptel.ac.in/courses/102103039/module3/lec17/3.html
 REPLICATION CYCLE

Structural
protein

Genomic RNA

Antigenome
Genome

Genome

Subgenome
Polyprotein 26S mRNA

Non-structural
protein

G.Antonelli e M.Clementi - Principi di Virologia Medica (2012)

 ALPHAVIRUS
INFECTION of INFECTION OCCASIONAL
RESERVOIR of VECTOR HOST

G.Antonelli e M.Clementi - Principi di Virologia Medica (2008)

Alphavirus pathogenetic for humans

http://www.google.com/patents/WO2010057141A2?cl=en
 ALPHAVIRUS
PATHOGENESIS:

G.Antonelli e M.Clementi - Principi di Virologia Medica (2008)

 ROSS RIVER VIRUS
➢ VECTOR: Mosquito ( Aedes and Culex)

➢ EPIDEMIOLOGY: Australia and Papua New Guinea

➢ INCUBATION: normally 7-9 days

➢ VIRUS STRAIN: different with different virulence

➢ In the 55-75% of cases infection in asymptomatic

 RRV
SYMTOMATOLOGY

❑ Low-grade fever (37,5-38,5 °C)

❑ Maculopapular rash on the trunk and limbs
(sometimes on the palms, the soles of feet and
face)
❑ Joint pain
❑ Stiffness and swelling that affects the wrists,
hands, fingers, ankles and kness

Additional manifestations may include:

❑ Head ache
❑ Diarrhea
❑ Nausea

The symptoms most often resolve within 3-6 months

 RRV
DIAGNOSIS

✓ Evidence of symptoms and clinical signs of disease to RRV

✓ Travel and stay in a RRV known or probable broadcast area

✓ Laboratory tests

Serology: IgM and IgG (IgM+ are highly suggestive but

seroconversion or a 4-fold increase of IgG titer is diagnostic of the
disease by RRV)

▪ RRV IgM may persist for months or years after the primary infection

▪ The first serum sample should be collected during the acute phase (within 7
days of onset of symptoms) and at least 10 days after the second sample
 RRV
TREATMENT

The support treatment includes:

✓ the use of non-steroidal anti-

inflammatory drugs
✓ the use of analgesics
✓ rest

VACCINE

Vaccination is not a useful approach because the attempts to develop a

vaccine for RRV have not been very successful on the duration of efficacy
of the vaccine and the ability to confer protective immunity against
different genotypes
 RRV
… IN EUROPE

The documented cases in Europe are associated with infected

travelers in Australia
 CHIKUNGUNYA
➢ VECTOR: Aedes spp. i.e. Mosquito ( Aedes aegypti and Aedes albopictus)

➢ ISOLATED: Tanzania 1952

➢ HOST: human

➢ GENOTYPE:
▪ African
▪ Asian
▪ East-central of South Africa (ECSA)
Medscape - PLoS Med (2006)
 CHIKV
TRANSMISSION CYCLE

➢ The main virus reservoirs are monkeys and other primates, mammals and
birds can be affected

Thiboutot MM et al., PLoS Negl Trop Dis (2010)

➢ The mosquito takes the virus from a person infected during the period of viremia
 CHIKV
EPIDEMIOLOGY

Outbreaks of Chikungunya virus is usually found in:

1.

1. Africa
2. Indian sub-continent and Indian Ocean islands
3. Southeast Asia

2
3
1
 CHIKV
EPIDEMIOLOGY
▪ Late 50s Thailand;
▪ 1960–1970 India;
▪ 1999 Port Klang in Malaysia;
▪ 2004 tropical and sub-tropical areas of Africa, Asia, Europe, the Pacific islands
and the Americas;
▪ 2005-2006 the island of La Reunion (French Republic);
▪ 2007 Italy;
▪ 2013 France;
▪ 2015 Latin America, the Caribbean and the United States of America

G. Rezza - Pathogens and Global Health (2014)

 CHIKV

➢ During the epidemic of 2005, there has been a change in the genetic
sequence allowing the virus to replicate more quickly inside the mosquito

➢ This mutation involves the substitution of alanine and valine amino-acid at

position 226 of the E1 protein (one of the major envelope glycoproteins) of
CHIKV (A226V)

➢ The change allows the virus to use the tiger mosquito (an invasive species)
as a carrier so could mean an increased risk of outbreaks in other areas
 CHIKV
… IN ITALY
➢ In summer 2007, there was an outbreak of infection in two small villages in
Castiglione di Cervia and Castiglione di Ravenna (Emilia Romagna)

➢ After the first outbreak, four secondary outbreaks have developed in Cervia,
Ravenna, Cesena and Rimini

A total of 248 cases

confirmed by laboratory
investigations

M.Calzolari - Eurosurveillance (2014)

➢ The isolated strain of CHIKV in Italy was found to be characterized by a

mutation in the E1 (E1: A226V)

➢ The vector of this outbreak is most likely the mosquito Aedes albopictus
(Asian tiger mosquito)
 CHIKV
CLINICAL COUSE

➢ The incubation period is 1-12 days, with a mean of 2-4 days

➢ The disease has a biphasic pattern typically:

6-10 days, characterized by fever, headache and arthralgia, which

I greatly limit the movements. Fever resolves after 4 days

2-3 days, characterized by the appearance of a maculopapular

II rash throughout the body and by the reappearance of the fever

➢ The majority of patients recovers completely, but a proportion of patients

develops arthritic symptoms that persist for several months or even years

➢ The elderly (> 65 years), and pediatric patients, especially infants, are
associated with a higher risk of complications and even death
 CHIKV
SYMPTOMATOLOGY

❑ High fever

❑ Arthralgia (severe joint pain)

❑ Marked weakness

❑ Cutaneous manifestations

❑ Muscle aches

❑ Headache

❑ Maculopapular rash of the trunk and

limbs

Potential complications include gastrointestinal complications, cardiovascular

failure or meningoencephalitis
 CHIKV
DIAGNOSIS

✓ Viral isolation

in 1-2 weeks

✓ RT-PCR

in 1-2 days

✓ Serological diagnosis

in 2-3 days
 CHIKV
TREATMENT
✓ No specific treatment

✓ The treatment is based on a supporting therapy with:

▪ Analgesics
▪ Antipyretics
▪ Antinfiammatory

➢ Movement and mild exercise tend to improve stiffness and morning

arthralgia, but heavy exercise may exacerbate rheumatic symptoms

CONTROL
The control activities are focused on:

✓ The use of pesticides to help eliminate mosquito;

✓ The use of insect repellents on exposed parts of the body

 CHIKV
VACCINE
➢ Currently a marketable vaccine is not available

➢ Many technologies have been used to develop vaccines, including

inactivated virus, live attenuated virus, alphavirus chimeras, DNA vaccines,
recombinant subunit vaccines and, more recently, using a virus-like particle
vaccine (VLP)

Scott C Weaver et al., Expert Rev Vaccines (2013)

 ENCEPHALITIS
VIRUS

VENEZUELAN ESTERN EQUIN WESTERN EQUIN

EQUIN ENCEPHALITIS ENCEPHALITIS
ENCEPHALITIS VIRUS (WEEV)
VIRUS (VEEV) VIRUS (EEEV)
 ENCEPHALITIS VIRUS
➢ Epidemiological cycles provide a sylvan transmission between mosquitoes
and birds, which act as a natural reservoir

➢ The infection can then be transmitted to the equine and human race

Pfeffer M, Parasit Vectors (2010)

➢ In infection by EEEV and WEEV, horses and humans are ''dead-end'' host
and do not develop a high viremia level

➢ In infection by VEEV, equines and humans act like guests amplification,

develop a high viremia level and are capable of transmitting the disease
through mosquitoes to other equines and humans
 EPIDEMIOLOGY

➢ Outbreaks of these viruses are usually found in America

N. Arèchiga-Ceballos et al. - Rev. Sci. Tech. Off. Int. Epiz (2015)

➢ VECTOR: Culex tarsalis

➢ ISOLATED: California 1930

➢ MORTALITY RATE: 3-7% in human

➢ INCUBATION: 2-10 days

➢ Vertical transmission

▪ In North America the last documented human case dates back to 1994
▪ In 2011 it was isolated a fatal human case in Uruguay
 WEEV
SYMPTOMATOLOGY
Mild disease with nonspecific symptoms:
❑ Temperature
❑ Nausea
❑ Vomit
❑ Anorexia
❑ Malaise
❑ Headache
❑ Respiratory symptoms (rare)

Other manifestations can include neurological symptoms:

❑ Restlessness
❑ Irritability
Especially in children
❑ Tremor
particularly under 1 year old
❑ Weakness
❑ Signs of meningeal irritation
➢ VECTOR: Culiseta Melanura

➢ ISOLATED Virginia e New Jersey 1933

➢ MORTALITY RATE: high mortality rate

➢ INCUBATION: 4-10 days

➢ VIRUS STRAIN: 4 genetic lines (I,II,IIIIV)

▪ I:North America
▪ II-III-IV: South America
 EEEV
SYMPTOMS

Initial symptoms:
❑ Sudden onset of fever
❑ Chills
❑ Myalgia
❑ Arthralgia
❑ Abdominal pains
❑ Headache

Often after a few days they are having characteristic neurological signs of encephalitis:

❑ Irritability
❑ Tremors
❑ Focal neurological deficit
❑ Convulsions
❑ Stiff neck
❑ Paralysis
❑ Confusion
❑ Respiratory symptoms
❑ Drowsiness
❑ Leukocytosis
❑ Disorientation
❑ Hematuria
 EEEV
COMPLICATIONS
➢ Children sometimes develop:
▪ Generalized edema
▪ Facial edema
▪ Peri-orbital edema
▪ Coma

➢ Infants may develop encephalitis without other symptoms

➢ Who does not develop neurological signs heals completely after a 1-2
week illness

➢ Only 10% of patients fully recover, and many survivors with severe die
within a few years

➢ The permanent neurological damage and death are particularly common in

children
➢ VECTOR: Mosquito ( Ochlerotatus taeniorhunchus,
during epidemics, and Culex, during zoonoses)

➢ ISOLATED: Venezuela 1938

➢ EPIDEMIOLOGY: mainly in Central and South America

➢ INCUBATION: normally 1-3 days, 6-7 for the most serious neurological
manifestations

➢ VIRAL STRAIN: 6 viral subtypes (I-VI)

▪ The subtype I is further divided into 5 antigenic veriants (AB-F)

 VEEV
SYMPTOMATOLOGY
➢ The rapid progression of clinical signs and the greater severity of the disease
differ from the VEE and WEE from EEE
➢ I Symptoms resolve within 1-2 weeks

BIPHASIC PERFORMANCE:
First phfase (about 48 hours):
❑ High fever
❑ Headache
❑ Loss of appetite
❑ Depression
❑ Macular rash
❑ Arthralgia
❑ Minor neurological signs (abnormal consciousness, drowsiness, reflex
excitability or lack of coordination)

Second phase:
❑ Normal temperatures
❑ Severe neurological symptoms
 VEEV
MORTALITY AND MORBODITY

➢ The mortality rate is below 1% in healthy adults

➢ The patients are young and the elderly are more likely to develop serious
illness

➢ 4 -15% of symptomatic patients may have mild neurological signs or

severe and may increase the mortality rate
 DIAGNOSIS ( WEEV, EEEV, VEEV)
✓ Serology: IgM and IgG specific in the cerebrospinal fluid (CSF)

IgM + are highly suggestive but seroconversion

or a 4-fold increase of IgG titer is diagnostic of
the disease

✓ RT-PCR

✓ Viral isolation

▪ VEEV: blood and swab oral (during the initial stage of the disease) and
CSF

▪ EEEV e WEEV: CSF of patients with neurological signs

 TREATMENT ( WEEV, EEEV, VEEV)

✓ No specific treatment

✓ Supportive therapy based on mechanical ventilation

VACCINE

EEEV: there are no commercial vaccine

WEEV: there are available for horses

VEEV: ▪ Strain C-84: to immunize horses

▪ Strain TC-83: available for at risk
individuals (military subjects, laboratory
technicians)
 Medically important bacteria

Flexible
Lacking cell walls Rigid cell walls
(spirochetes)
Mycoplasma Borrelia
Ureaplasma Leptospira
Treponema
Obligate intracellular
Filamentous Free-living
parasite

Actinomyces Chlamydia
Mycobacterium Rickettsia
Nocardia Coxiella
Gram positive Gram negative

Cocci (aerobic) Rods

Cocci Rods
Neisseria Aerobic Anaerobic
Aerobic Anaerobic Aerobic Anaerobic
Bacteroides
Fusobacterium
Staphylococcus Peptostreprococcus Corynebacterium Clostridium Enterobacteriaceae Others
Streptococcus Listeria Bacillus
Campylobacter Acinetobacter
Enterococcus Lactobacillus Propionibacterium Enterobacter
Escherichia Bordetella
Fusobacterium Brucella
Helicobacter Haemophilus
Klebsiella Legionella
Prevotella Moraxella
Proteus
Pseudomonas
Salmonella
Serratia
Shigella
Vibrio
Aeromonas
Yersinia
Phylum Spirochaetae Garrity
and Holt, 2001
Class Spirochaetes Cavalier-Smith,
2002
Direct Children:
Order Spirochaetales Buchanan, 1917
The bacteria in the order Spirochaetales have been grouped together on the basis of their
common morphologic properties.
• Thin, helical, gram negative bacteria

• 4 families and 14 genera

• Three genera responsible for human

diseases
 The most important treponemal species that causes human
disease is Treponema pallidum, with three subspecies. The
 subspecies are distinguished by their epidemiologic
characteristics, clinical presentation, and host range in
experimental animals. T. pallidum subspecies pallidum
(referred to as T. pallidum in this chapter) is the etiologic agent
of the venereal disease syphilis; T. pallidum subspecies
endemicum causes endemic syphilis (bejel); and T. pallidum
subspecies pertenue causes yaws. Bejel and yaws are
nonvenereal diseases.

 T. pallidum and related pathogenic treponemes are thin,

tightly coiled spirochetes (0.1 to 0.2 × 6 to 20 μm) with
pointed, straight ends.

 Tricarboxylic acid cycle is missing in the bacteria and they

are dependent on host cells for all purines, pyrimidines, and
most amino acids. In addition, spirochetes are microaerophilic
or anaerobic and extremely sensitive to oxygen, consistent
with the discovery the bacteria have no genes for catalase or
superoxide dismutase to protect them from oxygen toxicity. -
> FAILURE TO GROW IN VITRO
 Natural History of Untreated
Syphilis. The time intervals
between stages of syphilis
are shown, along with the
approximate percentages of
persons progressing to the
indicated stages. Invasion of
the central nervous system
(CNS) by treponemes may
not be a necessary
prerequisite for the
development of certain forms
of ocular syphilis. Adapted
from Ho and Lukehart.
Penicillin is the drug of choice for treating T. pallidum infections. A single intramuscular dose of long-acting
benzathine penicillin G is used for the early stages of syphilis, and three doses at weekly intervals is
recommended for congenital and late syphilis.

Doxycycline or azithromycin can be used as alternative antibiotics for patients allergic to penicillin.

Only penicillin can be used for the treatment of neurosyphilis. This is also true for pregnant women, who
should not be treated with tetracyclines.

Treatment failures with macrolides have been observed, so patients treated with azithromycin should be
closely monitored.

Because protective vaccines are not available, syphilis can be controlled only through the practice of safe-
sex
techniques and adequate contact and treatment of the sex partners of patients who have been
documented with infection.
Members of the genus Borrelia stain poorly with the Gram stain
reagents and are considered neither gram positive nor gram
negative, even though they have an outer membrane similar to
gram-negative bacteria.

Members of the genus Borrelia cause two important human

diseases: Lyme disease and relapsing fever.

They are larger than other spirochetes (0.2 to 0.5 × 8 to 30 μm),

stain well with aniline dyes (e.g., Giemsa or Wright stain), and can
be easily seen by light microscopy when present in smears of
peripheral blood from patients with relapsing fever but not those
with Lyme disease (too few organisms to be observed).

Borreliae are microaerophilic and have complex nutritional needs

(i.e., requiring N-acetylglucosamine, long-chain saturated and
unsaturated fatty acids, glucose, amino acids), which makes them
difficult to grow in vitro. Because culture is generally
unsuccessful, diagnosis of diseases caused by borreliae is by
serology (Lyme disease) or microscopy (relapsing fever).
Lyme disease is a tick-borne disease with protean
Relapsing fever is a febrile illness characterized by recurrent
manifestations, including dermatologic, rheumatologic,
episodes of fever and septicemia separated by
neurologic, and cardiac abnormalities. Initially it was
afebrile periods. Two forms of the disease are recognized.
believed that all cases of Lyme disease (or Lyme
B. recurrentis is the etiologic agent of epidemic or louse
borreliosis) were caused by one organism, B.
borne relapsing fever and is spread person to person by
burgdorferi.
the human body louse (Pediculus humanus). Endemic
However, subsequent studies have determined that a
relapsing fever is caused by as many as 15 species of
complex of at least 10 Borrelia species are responsible
borreliae and is spread by infected soft ticks of the genus
for Lyme disease in animals and humans.
Ornithodoros
Three species, B. burgdorferi, B. garinii, and Borrelia
afzelii, cause human disease, with B. burgdorferi found
in the United States and Europe and B. garinii and B.
afzelii found in Europe and central to eastern Asia.
The enzootic cycle of Borrelia burgdorferi
Ixodes spp. ticks
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The stages and most common clinical features
of Lyme borreliosis.
 Microscopic examination of blood or tissues from patients with Lyme disease is not recommended because B.
burgdorferi is rarely seen in clinical specimens. Borreliae that cause relapsing fever can be observed during the
febrile period on Giemsa-stained or Wright-stained preparation of blood. This is the most sensitive method for
diagnosing relapsing fever, with smears positive for organisms in more than 70% of patients.
 Some borreliae, including B. recurrentis and Borrelia hermsii (a common cause of endemic relapsing fever in the
United States), can be cultured in vitro on specialized media. The cultures are rarely performed in most clinical
laboratories because the media are not readily available, and the organisms grow slowly on them.
 Nucleic acid amplification techniques have a sensitivity of approximately 65% to 75% with skin biopsies, 50% to
85% with synovial fluid, and 25% with CSF specimens from patients with documented Lyme disease.
 Serologic tests are not useful in the diagnosis of relapsing fever because the borreliae that cause this condition
undergo antigenic phase variation. In contrast, serologic testing is the diagnostic test of choice for patients with
suspected Lyme disease. The tests most commonly used are the immunofluorescence assay (IFA) and EIA.
The U.S.
 More than 70 serologic assays for the diagnosis of Lyme disease. Unfortunately, all serologic tests are relatively
insensitive during the early acute stage of disease. IgM antibodies appear 2 to 4 weeks after the onset of
erythema migrans in untreated patients; the levels peak after 6 to 8 weeks of illness and then decline to a
normal range after 4 to 6 months. The IgM level may remain elevated in some patients with a persistent
infection. The IgG antibodies appear later. Their levels peak after 4 to 6 months of illness and persist during the
late manifestations of the disease
 The early manifestations of Lyme disease are managed effectively with orally
administered amoxicillin, doxycycline, or cefuroxime
 Relapsing fever has been treated most effectively with tetracyclines or penicillins.
Tetracyclines are the drugs of choice but are contraindicated for pregnant women and
young children. A Jarisch-Herxheimer reaction (shocklike profile with rigors, leukopenia,
an increase in temperature and a decrease in blood pressure) can occur in patients
within a few hours after therapy is started and must be carefully managed. This reaction
corresponds to the rapid killing of borreliae and the possible release of toxic
products.and high-risk sexual practices in homosexual males.
 Vaccines are not available for relapsing fever. A recombinant vaccine directed against
the OspA antigen of B. burgdorferi was removed from the market in 2002.
 To read (not exam program)

 Mycoplasma&Ureaplasma_Section8ClinicalMicrobiology
 SECTION 8 Clinical Microbiology: Bacteria

186
Mycoplasma and Ureaplasma
JØRGEN SKOV JENSEN

KEY CONCEPTS Epidemiology

• Mycoplasmas are primarily mucosal pathogens, but some M. pneumoniae causes mild upper respiratory tract symptoms more
species may cause invasive infections in susceptible hosts. often than severe disease, and infections have been described from
most parts of the world. In temperate climates, most infections are
• Mycoplasma pneumoniae causes mild upper respiratory tract reported during late summer until mid-winter. However, the infection
symptoms but is a major cause of community acquired pneu-
is endemic throughout the year with epidemic peaks about every 4–7
monia in older children and young adults. Macrolide resistance
is spreading and in some areas is nearly 100% (Asia) but is years.1 The incubation period ranges from 2 to 4 weeks. Spread from
considerably lower in Europe and in the USA. person to person occurs slowly, usually where there is continual or
repeated close contact, as within a family.2,3
• Mycoplasma genitalium is an emerging sexually transmitted M. pneumoniae is only responsible for a minority of all upper tract
infection and an established cause of acute and persistent or infections, where viral infection is the dominating cause, but it is a
recurrent non-gonococcal urethritis. Macrolide resistance is
significant cause of lower respiratory tract infections being reported in
spreading and alternative treatment with moxifloxacin is threat-
ened, leaving very few options for treatment. 15–20% of all pneumonias in the USA.4 In closed communities such
as military camps and institutions, outbreaks with a high attack rate
• Mycoplasma hominis and ureaplasmas are common urogenital have been reported.3
commensals, but may cause disease in a susceptible host.
• Ureaplasma urealyticum but not U. parvum may cause male Pathogenicity
non-gonococcal urethritis when present in high concentrations. Infection with M. pneumoniae probably happens in all age groups but
• Mycoplasma hominis and ureaplasmas should be considered the risk of developing pneumonia varies with age. Approximately 25%
in wound infections after urogenital tract surgery or after of children 5–15 years old with M. pneumoniae infection develop
transplantation. pneumonia, whereas this is the case in only about 7% of younger
adults, although they often experience milder disease. Pneumonia is
less frequent thereafter, but is more severe in older patients.5
A crucial step in M. pneumoniae infection is the adherence of the
Introduction organisms to respiratory mucosal epithelial cells, which is mediated by
a specialized terminal tip-structure carrying the P1 main adhesion
The class Mollicutes (meaning ‘soft skin’), trivially known as mycoplas- protein and accessory proteins. After attachment to the ciliated epithe-
mas, comprise some of the smallest free-living micro-organisms. They lium, M. pneumoniae secretes hydrogen peroxide leading to ciliostasis,
lack the cell wall found in most other bacteria and, consequently, they and subsequent necrosis of the epithelium.6 The pneumonia is mainly
are resistant to β-lactam antimicrobials. The small size of the myco- caused by peribronchiolar and perivascular pulmonary infiltration
plasma genome limits the metabolic capabilities, making culture of with lymphocytes and is mainly an immune-pathological process since
some mycoplasmas difficult or impossible. Mycoplasmas isolated com- immunosuppression prevents pneumonia or diminishes its severity.7
monly from humans belong to the genera Mycoplasma and Ureaplasma
within the order Mycoplasmatales. The genus Ureaplasma (‘ureaplas-
mas’) is unique in the ability to hydrolyze urea.
Prevention
The Mollicutes are also economically important plant and animal No vaccine against M. pneumoniae has been developed, and early
pathogens. Although most species have strict host specificities, some attempts with vaccination resulted in more severe disease. Azithromy-
of the animal mycoplasmas may occasionally cause infections in cin treatment has been used successfully to prevent spread of epidem-
humans. ics in confined settings, but is not generally recommended.8
Currently, 14 Mycoplasma species and two Ureaplasma species are
considered human mycoplasmas (summarized in Table 186-1). In Diagnostic Microbiology
this chapter, only the five species of clinical significance will be Diagnosis can be made by molecular methods such as NAAT, serology
considered: and culture. Traditionally, serology has been the primary diagnostic
• Mycoplasma pneumoniae method, but this is increasingly being replaced by molecular methods.
• Mycoplasma genitalium NAATs such as polymerase chain reaction (PCR) are capable of provid-
• Mycoplasma hominis ing rapid diagnostic results in the acute phase of the disease, where it
• Ureaplasma urealyticum may direct antimicrobial treatment. Various gene targets have been
• Ureaplasma parvum. used, but no standardization or general recommendations exist.9
Recently, commercially available detection kits have received US Food
and Drug Administration (FDA) approval.
Mycoplasma pneumoniae Serology is generally less costly than NAATs, but since antibodies
peak at 3–4 weeks after onset of disease, this method may have less
Nature relevance in the acute phase of the disease. Commercially available
Mycoplasma pneumoniae is an important respiratory tract pathogen. It enzyme immunoassays specific for IgG and/or IgM are commonly
grows slowly in specialized bacteriological media, and infection is pri- used. IgM detection is much less reliable in re-infection, which is most
marily diagnosed by nucleic acid amplification tests (NAAT), or indi- often the case in adults. Cold agglutinins, detected by agglutination of
rectly by serology. O Rh-negative erythrocytes at 4 °C, correlate with specific IgM and are
1660
 Chapter 186 Mycoplasma and Ureaplasma 1661

TABLE Mycoplasma and Ureaplasma Species Considered of Human Origin, Their Preferred Colonization and
186-1 Disease Association
DETECTION RATE

Species Respiratory Tract Genitourinary Tract Rectum Blood Cause of Disease

M. amphoriforme Rare —* —* —* Possibly

M. buccale Rare — — — No

M. faucium Rare — — — No

M. fermentans Common Rare — Very rare Possibly

M. genitalium Rare Common Rare ? Yes

M. hominis Rare Common Common Rare Yes

M. lipophilum Rare — — — No

M. orale Common — — — No

M. penetrans — Rare Very rare ? ?

M. pirum ? — Rare Very rare ?

M. pneumoniae Rare† Very rare — — Yes

M. primatum — Rare — — No

M. salivarium Common Rare — — No

M. spermatophilum — Rare — ? ?

U. parvum Rare Common Common Very rare Yes

U. urealyticum Rare Common Common Very rare Yes

*No reports of detection.

†
Except in disease outbreaks.

suggestive of a recent M. pneumoniae infection, but have a low specific- can be precipitated by cold agglutinins, which are autoimmune anti-
ity and the test is rarely used.10 bodies against the I-antigen on erythrocytes. Cross-reacting autoanti-
Complex culture media are needed for isolation of M. pneumoniae, bodies may be responsible for neurological and other complications,
and as culture may take up to 5 weeks, it is of no clinical value. It does, although direct invasion of the central nervous system may explain a
however, allow for antimicrobial susceptibility testing and is therefore small proportion of cases. Skin manifestations such as an urticarial
important in surveillance and research.11 rash are quite common and may be misinterpreted as allergic reactions
to the commonly prescribed macrolide treatment. Stevens–Johnson
Clinical Manifestations syndrome is often associated with M. pneumoniae infection (Table
M. pneumoniae produces a range of clinical manifestations from 186-2). Mycoplasma has been thought of as a cause of bullous myrin-
asymptomatic infection and mild, afebrile, upper respiratory tract gitis but more recent studies suggest this is a false association.
disease to severe pneumonia. The most typical clinical syndrome is
tracheobronchitis, often accompanied by upper respiratory tract
symptoms, such as acute pharyngitis. M. pneumoniae pneumonia
Management
cannot be distinguished from other etiologies without laboratory M. pneumoniae is generally susceptible to tetracyclines and macrolides.
testing.12 Illness often starts with a few days of malaise and headache Ciprofloxacin and similar quinolones have only moderate activity,
before respiratory symptoms appear, and pneumonia is seen radio- whereas the newer quinolones, such as moxifloxacin, are highly active
graphically before physical signs, such as rales, are detectable. Usually, in vitro. They should not be used as first-line therapy but may have a
only one of the lower lobes is involved and the radiograph shows role in immunosuppressed patients, as they are bactericidal. High-level
patchy opacities but approximately 20% of patients have bilateral macrolide resistance in M. pneumoniae has become extremely common
pneumonia. Pleuritis and pleural effusions are unusual. Illness is often in some areas, with nearly 95% resistance reported among infected
protracted, and symptoms may persist for several weeks, and may patient in parts of Asia.14 However, only 1–10% resistant strains have
relapse. M. pneumoniae can persist in respiratory secretions for weeks been reported from Europe and the United States.15
despite antibiotic therapy and apparent clinical cure. Mortality is low, Tetracyclines and macrolides significantly reduce the duration of
although patients with immunodeficiency or sickle cell anemia may fever, pulmonary infiltration and other signs and symptoms, and treat-
experience severe infections. In children, illness may be prolonged with ment with an active antimicrobial is recommended. In areas with low
paroxysmal cough followed by vomiting, simulating whooping cough. levels of macrolide resistance, azithromycin is commonly used, often
M. pneumoniae has been implicated in bronchial asthma, but this is as 500 mg once daily for 3 days.
controversial.

EXTRAPULMONARY MANIFESTATIONS OF Mycoplasma genitalium

M. PNEUMONIAE INFECTION
Infections with M. pneumoniae are occasionally associated with various Nature
extrapulmonary symptoms which may occur simultaneously with the Mycoplasma genitalium is an emerging sexually transmitted infection
respiratory symptoms or after these have resolved.13 Hemolytic crisis and the bacterium is very closely related to M. pneumoniae. Indeed, all
1662 SECTION 8 Clinical Microbiology: Bacteria

in many samples.22,23 From men, the optimal diagnostic sample appears

TABLE Extrapulmonary Manifestations of to be first-void-urine (FVU), whereas in women, a combination of
186-2 M. pneumoniae Infections FVU and cervical or vaginal swabs detects most infections.24 As men-
tioned below, macrolide resistance is a major problem in some popula-
Estimated
System Manifestation Frequency
tions, and rapid NAAT-based detection of macrolide resistance
mediating mutations has been introduced in a few laboratories to
Dermatological Urticaria, erythema multiforme, Some guide treatment.25
Stevens–Johnson syndrome, other involvement
rashes in about 25%

Gastrointestinal Anorexia, nausea, vomiting and 14–44% Clinical Manifestations

transient diarrhea
DISEASE IN MEN
Hepatitis ?
Acute Non-gonococcal Urethritis
Pancreatitis ? M. genitalium was isolated initially from men with acute NGU and
Musculoskeletal Myalgia, arthralgia, arthritis 14–45% more than 35 studies have shown M. genitalium to be strongly and
almost uniformly associated with acute NGU.17 Among STD clinic
Genitourinary Acute glomerulonephritis Insignificant populations, about 90% of M. genitalium-infected men have micro-
Hematological Cold agglutinin production About 50% scopic evidence of urethritis and 70–80% report symptoms. The asso-
ciation between M. genitalium and urethritis is even stronger in acute
Hemolytic anemia ?
non-chlamydial NGU,17 where it accounts for more than one-third of
Thrombocytopenia ? the cases, strongly suggesting that M. genitalium and C. trachomatis act
Intravascular coagulation >50 reported
as separate causes of the condition.
cases
Chronic Non-gonococcal Urethritis
Neurological Meningitis, meningoencephalitis, 6–7% in a few M. genitalium is found in up to 40% of men with persistent or recur-
ascending paralysis, transient studies based
myelitis, cranial nerve palsy, on serology
rent NGU after treatment with doxycycline,26,27 probably due to the
poliomyelitis-like illness, Guillain– inefficiency of tetracyclines in eradicating M. genitalium.
Barré syndrome
Chronic Prostatitis
Cardiovascular Myocarditis, pericarditis <5%
Despite M. genitalium being involved in chronic NGU, there is only
limited evidence suggesting that it is associated with chronic
prostatitis.

M. genitalium genes present in the very small genome (580 kbp) can
Acute Epididymitis
be found in the larger (816 kbp) M. pneumoniae genome.16 Clinical experience as well as the detection of M. genitalium in a few
patients during an antibiotic trial28 suggests that M. genitalium may be
Epidemiology a cause of acute epididymitis in some patients. In a study from Japan
it was detected in 9% of men <40 years of age with epididymitis,29 but
M. genitalium is strongly associated with acute non-gonococcal ure- often together with C. trachomatis.
thritis (NGU) in both men and women. It is detected in approximately
25% of cases but in only 5–10% of asymptomatic sexually transmitted Proctitis
disease (STD) clinic attendees and in 1–3% of asymptomatic healthy M. genitalium has been detected in 2–11% of rectal swabs from men
controls.17 In symptomatic men it has been detected almost indepen- who have sex with men (MSM), but the relation to symptoms is not
dently of Chlamydia trachomatis.18 In both men and women, the clear.30 Whether a rectal infection with M. genitalium can be transmit-
highest prevalence is found in individuals slightly older than those with ted, remains to be determined.
C. trachomatis.19
DISEASE IN WOMEN
Pathogenicity Non-gonococcal Urethritis
Similar to M. pneumoniae, M. genitalium has a specialized terminal M. genitalium has been associated with urethritis in women attending
structure mediating cell-adherence. STD clinics.31,32 It is unclear if M. genitalium explains a proportion of
Experimentally, M. genitalium causes urethritis in male chimpan- sterile pyuria.
zees,20 and adheres to and enters epithelial cells.21 Intracellular M.
genitalium may be partially protected from antimicrobials, resulting in Bacterial Vaginosis (BV) and Vaginitis
persistent or recurrent non-gonococcal urethritis. The association of M. genitalium with BV is controversial; most studies
have not shown an association; M. genitalium has not been associated
Prevention with vaginitis.
No vaccine against M. genitalium is available, but as with other sexually
Cervicitis
transmitted infections, consistent use of condoms provides good
protection. M. genitalium is generally detected in 10–20% of women with cervici-
tis, and in the majority of studies, significantly more often in cases
than in controls.30 The association between M. genitalium and cervici-
Diagnostic Microbiology tis is less strong than that seen for male NGU with pooled odds-ratios
Culture of M. genitalium is extremely difficult and time-consuming around 2.2.17
and is not useful for diagnosis. Serology is severely hampered by cross-
reactions with pre-existing M. pneumoniae antibodies, which are Pelvic Inflammatory Disease (PID)
present in most adult patients. Consequently, NAATs are the only PID is an inflammation of the upper genital tract and comprises endo-
useful diagnostic methods, but at present, no commercially available metritis and/or salpingitis. For all NAAT-based studies, M. genitalium
FDA approved test exists. Test performance may vary significantly has been associated with PID.17,30 Odds-ratios for the association have
between laboratories due to the low amount of M. genitalium present been from 2.1 to 6.3, and symptom severity has been comparable to
 Chapter 186 Mycoplasma and Ureaplasma 1663

that experienced in chlamydial PID, but milder than for gonococcal Epidemiology
PID.33 In most studies, 5–15% of PID could be attributed to M. geni-
talium, suggesting that in many settings it would significantly outnum- Ureaplasmas and M. hominis can be transmitted to about 40% of
ber cases caused by N. gonorrhoeae. babies born to infected mothers.51 Infants above 3 months of age are
Importantly, M. genitalium was also strongly associated with PID rarely colonized.52
after termination of pregnancy, an observation that calls for further In adults, the rate of colonization increases with the number of
studies, as screening before the procedure as for C. trachomatis might different sexual partners.53 M. hominis and ureaplasmas can be found
be important.34 in the cervix or vagina of 20–50% and 40–80% of sexually active,
asymptomatic women, respectively. The colonization rate is somewhat
Infertility lower in the urethra of healthy males. It is important to note that colo-
Serological studies have shown an association with tubal factor infertil- nization is strongly linked to BV in women. This may lead to an over-
ity with around 20% of women having M. genitalium antibodies com- estimation of the importance of M. hominis and ureaplasmas in
pared to 5% with normal tubes.35,36 The association was statistically diseases where BV is a risk factor.
significant, also when controlling for C. trachomatis.
Pathogenicity
DISEASE IN BOTH MEN AND WOMEN
Although M. hominis adheres to cells and possesses several adhesins,
Sexually Acquired Reactive Arthritis many of which are immunogenic in the host, and subject to variation
There are no systematic studies of the role of M. genitalium in reactive in the bacterium,54 the adhesion is less strong than that seen in M.
arthritis and Reiter’s syndrome, but clinical experience suggests that it pneumoniae and M. genitalium. M. hominis metabolizes arginine, and
may have a role. M. genitalium has been detected by PCR in the knees releases ammonia, which may cause local tissue damage. Similarly, the
of a patient with Reiter’s syndrome.37 ureaplasmas adhere to cells55 and also produce ammonia from the
degradation of urea.
HIV and Immunosuppression
According to a meta-analysis, individuals infected with M. genitalium
are twice as likely to be infected with human immunodeficiency virus Diagnostic Microbiology
(HIV).38–40 M. genitalium is also associated with HIV shedding in M. hominis and ureaplasmas are so commonly found in the lower
women.41 genital tract of healthy men and women that detection from these sites
is rarely relevant. Ureaplasmas and M. hominis usually show evidence
of growth in special culture media within 1–5 days. M. hominis may
Management grow on ordinary blood agar where it produces pinpoint colonies after
Antimicrobial treatment of M. genitalium has become a complicated extended incubation. NAAT assays for detection of M. hominis and U.
issue. Although tetracyclines are active in vitro,42 they only eradicate urealyticum/U. parvum are available in some laboratories, and the
the infection from less than a third of infected patients. Azithromycin ability to distinguish the two Ureaplasma species is important. Quan-
in a 1 g single dose commonly used for C. trachomatis infection had titative assays are recommended over qualitative assays. Serology
cure rates around 85% in clinical trials.43,44 Azithromycin 500 mg day cannot be recommended for diagnostic purposes.
one followed by 250 mg days 2–5 as used for M. pneumoniae infections
had a cure rate of 95%.44 Unfortunately, an increasing proportion of
M. genitalium strains have developed resistance to macrolides through Clinical Manifestations
mutations in region V of the 23S ribosomal RNA gene.45 Rates of DISEASE IN MEN
macrolide resistance around 40% have been reported both in Australia Non-gonococcal Urethritis
and Europe,19,46,47 but in selected populations, resistance as high as The role of ureaplasmas in NGU is less clear than for M. genitalium.
100% has been reported.48 If azithromycin treatment fails, moxifloxa- Human inoculation studies56,57 support a causal role for ureaplasmas
cin (400 mg once daily for 7 days) can be used,49 but this agent is not in NGU, particularly chronic disease.58 The role of U. urealyticum is
recommended as first-line therapy. Unfortunately, increasing quino- still controversial, but an increasing number of studies have associated
lone resistance is seen, with emergence of strains resistant to both this species with NGU,59 in particular when present in high titers.60,61
azithromycin and moxifloxacin.42 Such strains may be extremely dif- U. parvum is detected more often from the control group, suggesting
ficult to eradicate, but limited clinical experience suggests that pris- that this species has a lower pathogenic potential. There is no evidence
tinamycin may be effective. supporting a role for M. hominis as a cause of urethritis.62
Prostatitis
Mycoplasma hominis and Ureaplasmas have been isolated from expressed prostatic secretions
after prostatic massage more often and in higher titers in men with
Ureaplasma spp. chronic prostatitis than in controls.63,64 However, there is not much
evidence to support a role in acute or in chronic bacterial prostatitis,
Introduction and M. hominis has not been associated with prostatitis of any kind.
In this section, M. hominis and the ureaplasmas will be described
together, as they are involved in the same disease conditions. The
Epididymitis
ureaplasmas have been reclassified from the original name U. urealyti- Ureaplasmas have been recovered from the urethra and directly from
cum to now comprise two separate species, U. urealyticum and U. epididymal aspirate fluid, accompanied by a specific antibody response
parvum.50 The two species cannot be distinguished by culture methods in a patient with acute epididymitis.65 However, further studies are
so species names will be used only where such distinction has been required to establish a causal role.
possible. In studies based on undifferentiated culture findings, the
DISEASE IN WOMEN
bacteria will be referred to as ureaplasmas.
Bacterial Vaginosis (BV)
BV affects 5–25% of women66 and is characterized by a loss of Lacto-
Nature bacillus species and an increase in gram-variable coccobacilli, anaero-
M. hominis and ureaplasmas are opportunistic pathogens, occasionally bic organisms, as well as M. hominis and ureaplasmas.67 Both
causing infections relating primarily to the urogenital tract. ureaplasmas and M. hominis are found in higher titers in the vagina of
1664 SECTION 8 Clinical Microbiology: Bacteria

women with BV than in healthy women, but they are not the cause of rarely been implicated in pneumonia soon after birth, but both M.
the condition.68 hominis and ureaplasmas have been isolated from the cerebrospinal
BV is a strong predictor of infectious complications in women, fluid of neonates with meningitis or brain abscess and should be con-
thus, the strong association between ureaplasmas, M. hominis, and BV sidered in culture-negative neonatal meningitis.85
leads to significant problems in defining the roles of these species, as
BV has not been controlled for in most studies. Postpartum and Postabortal Fever. M. hominis is considered to
be responsible for some cases of maternal fever after a normal delivery
Cervicitis or abortion, accounting for almost 10% of positive cultures.86 It is
important to note that most commercially available blood culture
The role of ureaplasmas and M. hominis in cervicitis has not been media contain sodium polyanetholesulfonate (SPS), which inhibits the
studied in great detail. Mucopurulent cervicitis has been associated growth of mycoplasmas;87 consequently, special media should be
with isolation of ureaplasmas,69 although the role of bacterial vaginosis employed when mycoplasma infection is suspected.
as a confounder was not assessed.

PID and Sequelae DISEASE IN MEN AND WOMEN

PID is caused by microbes in the lower genital tract invading the upper Urinary Tract Disease
genital tract. C. trachomatis and Neisseria gonorrhoeae are well estab- Pyelonephritis and Urinary Tract Infection. M. hominis does
lished etiological agents, but the mixed bacterial flora associated with not appear to play an important role in acute cystitis.88 However, urea-
BV is also a cause of PID,70 and the role of ureaplasmas and M. hominis plasmas have been associated with symptoms of cystitis and with the
should be considered in that context. M. hominis has been recovered acute urethral syndrome in women without significant bacteriuria, and
more frequently from lower tract specimens from women with PID have been isolated from up to 25% of suprapubic aspirates from such
than from healthy women.71,72 M. hominis has been isolated apparently women.89,90
in pure culture from the fallopian tubes of women with salpingitis Despite the lack of evidence for M. hominis causing acute cystitis,
diagnosed by laparoscopy, but not from women without lesions.73 In it has been found in up to 10% of cases of acute pyelonephritis.91 There
several studies, a fourfold rise in antibody titer to M. hominis has been is no evidence suggesting a similar role for ureaplasmas.
found more often among women who had PID than among controls,
but no control for BV was reported.71,74 Although ureaplasmas have Urinary Calculi and Catheter Encrustations. Infection stones
also been isolated occasionally from the fallopian tubes of patients with and catheter encrustations may be caused by urea-hydrolyzing bacteria
PID,75,76 they have usually been found in association with other known and ureaplasmas have been detected in 12–27% of infection stones,92
pathogens and are not thought to play a major role. more often in the urine and stones of patients with infection stones,
In conclusion, there is some evidence that M. hominis may be a compared to those with metabolic stones.93
cause of PID, possibly as part of the BV-associated flora, but there is Infections in Immunodeficient Patients
very little evidence that ureaplasmas have a similar role.
Patients with antibody deficiencies such as hypo- or agammaglobulin-
emia are particularly susceptible to extragenital mycoplasma infec-
Pregnancy-Related Complications tions. M. hominis and ureaplasmas have been found in septic arthritis,
Infertility. Although ureaplasmas and M. hominis have been associ- osteomyelitis, subcutaneous abscesses and cellulitis.94 Cystitis and
ated with both male-factor and female-factor infertility in some chronic urethritis is common and it is often very difficult to eradicate
studies, no convincing evidence has been obtained. the mycoplasmas.95,96 Patients undergoing organ transplantation are
Adverse Pregnancy Outcome. Isolation of ureaplasmas and M. also susceptible to both wound and systemic mycoplasma infections;
hominis from the lower genital tract have shown a only a weak associa- patients having kidney, lung, heart and liver transplantations appear
tion with spontaneous abortion compared to studies based on detec- to be at high risk.
tion from the endometrium or placenta but they may just be markers Prolonged intravenous and combination therapy with high doses
of concomitant BV, and BV in itself has been associated with both of antibiotics should be considered to avoid development of antimi-
pre-term labor and stillbirth.77,78 crobial resistance. In general, fluoroquinolones with extended gram-
Ureaplasmas have been isolated more frequently from spontane- positive spectrum such as moxifloxacin are recommended due to their
ously aborted fetuses and stillborn or premature infants than from bactericidal effect and potency against a broad spectrum of mollicutes,
induced abortions or normal full-term infants. Ureaplasmas have been but always in combination with another antibiotic class such as
isolated from lungs, brain, heart and viscera, suggesting that it was not tetracyclines.
merely superficial contamination.79,80
Chorioamnionitis and mycoplasma infection are significantly asso-
ciated, even when corrected for the duration of rupture of the mem- Management
branes. Both ureaplasmas and M. hominis can invade the amniotic sac Before treating M. hominis or ureaplasmas, the first consideration is
before 20 weeks of gestation in the presence of intact fetal membranes. whether to treat or not. The high carriage rate among healthy adults
Detection in mid-trimester amniotic fluid was associated with preterm should always be kept in mind.
premature rupture of the membranes with subsequent preterm The main antibiotics are the tetracyclines, the macrolides, quino-
birth.81,82 However, not all infected women delivered preterm. lones and clindamycin. M. hominis is intrinsically resistant to macro-
Neonatal Infections. Ureaplasmas can be isolated from the respi- lides but most strains are susceptible to tetracyclines, quinolones and
ratory tract of neonates. The isolation rate correlates strongly with clindamycin. Ureaplasmas are susceptible to tetracyclines, quinolones
birth-weight and infants below 1000 g are infected much more often and macrolides, whereas clindamycin is mostly inactive.
than full-term infants.83 A meta-analysis has shown that Ureaplasma
colonization of the lower respiratory tract of infants <1500 g increases References available online at expertconsult.com.
the risk of development of chronic lung disease.84 M. hominis has very
 Chapter 186 Mycoplasma and Ureaplasma 1665

KEY REFERENCES
Cassell G.H., Waites K.B., Crouse D.T., et al.: Association Jensen J.S., Bradshaw C.S., Tabrizi S.N., et al.: Azithromy- Taylor-Robinson D., Csonka G.W., Prentice M.J.: Human
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Eschenbach D.A., Buchanan T.M., Pollock H.M., et al.: 47(12):1546-1553. from chrysalis to multicolored butterfly. Clin Microbiol
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Foy H.M., Grayston J.T., Kenny G.E., et al.: Epidemiology 27(4):779-792. nancy. BJOG 2011; 118(2):164-174.
of Mycoplasma pneumoniae infection in families. JAMA McCormack W.M., Almeida P.C., Bailey P.E., et al.: Sexual Waites K.B., Katz B., Schelonka R.L.: Mycoplasmas and
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 Chapter 186 Mycoplasma and Ureaplasma1665.e1

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Bacillaceae
 Medically important bacteria

Flexible
Lacking cell walls Rigid cell walls
(spirochetes)
Mycoplasma Borrelia
Ureaplasma Leptospira
Treponema
Obligate intracellular
Filamentous Free-living
parasite

Actinomyces Chlamydia
Mycobacterium Rickettsia
Nocardia Coxiella
Gram positive Gram negative

Cocci (aerobic) Rods

Cocci Rods
Neisseria Aerobic Anaerobic
Aerobic Anaerobic Aerobic Anaerobic
Bacteroides
Fusobacterium
Staphylococcus Peptostreprococcus Corynebacterium Clostridium Enterobacteriaceae Others
Streptococcus Listeria Bacillus
Enterococcus Lactobacillus Propionibacterium Campylobacter Acinetobacter
Enterobacter Helicobacter Bordetella
Escherichia Brucella
Klebsiella
Fusobacterium
Prevotella Haemophilus
Salmonella Legionella
Shigella Proteus
Moraxella
Pseudomonas
Vibrio
Yersinia
Serratia
Phylum Firmicutes corrig. Gibbons and
Murray, 1978
Class Bacilli Ludwig et al., 2010
Order Bacillales Prévot, 1953
Family Bacillaceae Fischer, 1895
Genus Bacillus Cohn, 1872
• Gram-positive (though some may appear gram-variable or
even gram-negative), rod-shaped organism

• Anaerobic growth

• Forms endospores (usually elliptical and subterminal, not

associated with swollen sporangium in B. anthracis, B.
cereus, or B. thuringiensis)

• Catalase positive, often motile (except B.

anthracis and Bacillus mycoides)
- Rapidly differentiates from Clostridium

• Culture: All Bacillus species grow on blood agar

• Spores can be generated by heat treatment

Bacillus spp. are ubiquitous
•Soil, water, and airborne dust
•Thermophilic (< 75°C) and psychrophilic (>5-8°C)
•Can flourish at extremes of acidity & alkalinity (pH 2 to 10)

 On blood agar
•Large, spreading, gray-white colonies, with irregular margins
• Many are beta-hemolytic (helpful in differentiating various Bacillus species from
B. anthracis)
• Spores seen after several days of incubation, but not typically in fresh clinical
specimens
Bacillus anthracis
Virulent B. anthracis carries genes for three toxin protein components on a large plasmid, pXO1.

The individual proteins, protective antigen (PA), edema factor (EF), and lethal factor (LF), are
nontoxic individually but form important toxins when structurally combined: PA plus EF forms edema
toxin, and PA plus LF forms lethal toxin. PA is an 83-kDa protein that binds to one of two receptors on
host cell surfaces that are present on many cells and tissues (e.g., brain, heart, intestine, lung, skeletal
muscle, pancreas, macrophages, ANTXR1 (also known as tumor endothelial marker-8 (TEM8), and
ANTXR2 (also known as capillary morphogenesis protein 2 (CMG2)).

After PA binds to its receptor, host proteases cleave PA, releasing a small fragment and retaining the 63-
kDa fragment (PA63) on the cell surface. The PA63 fragments self-associate on the cell surface, forming
a ring-shaped complex of seven fragments (pore precursor or “prepore”).

This heptameric complex can then bind up to three molecules of LF and/or EF. Both factors recognize
the same binding site of PA63, so the binding is competitive. Formation of the complex stimulates
endocytosis and movement to an acidic intracellular compartment.
In this environment, the heptameric complex forms a transmembrane pore and releases LF and EF into the cell
interior.

LF is a zinc-dependent protease capable of cleaving mitogen-activated protein (MAP) kinase, leading to cell
death.
EF is a calmodulin-dependent adenylate cyclase that increases the intracellular cyclic adenosine
monophosphate (cAMP) levels and results in edema. EF is related to the adenylate cyclases produced by
Bordetella pertussis and Pseudomonas aeruginosa.

The other important virulence factor carried by B. anthracis is a prominent polypeptide capsule (consisting of
poly-D-glutamic acid). This protein capsule is unique because most bacterial capsules are composed of
polysaccharides (e.g., such as those on Staphylococcus aureus, Streptococcus pneumoniae, and P.
aeruginosa). The capsule is observed in clinical specimens, but it is not produced in vitro unless special growth
conditions are used.

Three genes (capA, capB, and capC) are responsible for synthesis of this capsule and are carried on a second
plasmid (pXO2). Only one serotype of capsule has been identified, presumably because the capsule is
composed of only glutamic acid.
Epidemiology
of Anthrax in
Animal and
Human Hosts
 Clinical presentation of Anthrax
Cutaneous Inhalation Gastrointestinal (Ingestion)
• 95% human cases are cutaneous • Virtually 100% fatal • Virtually 100% fatal
infections (pneumonic) • Abdominal pain
• 1 to 5 days after contact • Meningitis may • Hemorrhagic ascites
• Small, pruritic, non-painful papule complicate cutaneous • Paracentesis fluid may
at inoculation site and inhalation forms of reveal gram-positive rods
• Papule develops into hemorrhagic disease
vesicle & ruptures • Pharyngeal anthrax
• Slow-healing painless ulcer • Fever
covered with black eschar • Pharyngitis
surrounded by edema • Neck swelling
• Infection may spread to lymphatics
w/ local adenopathy
• Septicemia may develop
• 20% mortality in untreated
cutaneous anthrax
 Treatment & Prophylaxis

• Treatment
• Penicillin is drug of choice
• Erythromycin, chloramphenicol acceptable alternatives
• Doxycycline now commonly recognized as prophylactic

• Vaccine
• Laboratory workers
• Employees of mills handling goat hair
• Active duty military members
• Potentially entire populace of U.S. for herd immunity
 Diagnosis
Anthrax is one of the few bacterial diseases in which organisms can be seen when peripheral blood is Gram stained.

The diagnostic difficulty is distinguishing B. anthracis from other members of the taxonomically related B. cereus group. A
preliminary identification of B. anthracis is based on microscopic and colonial morphology. The organisms appear as long, thin,
gram-positive rods arranged singly or in long chains. Spores are not observed in clinical specimens; they are only seen in cultures
incubated in a low CO2 atmosphere and can be seen best with the use of a special spore stain (e.g., malachite green stain).
The capsule of B. anthracis is produced in vivo but is not typically observed in culture. The capsule can be observed in
clinical specimens using a contrasting stain, such as India ink (the ink particles are excluded by the capsule so that the
background, but not the area around bacteria, appears black), M’Fadyean methylene blue stain, or a direct fluorescent
antibody (DFA) test developed against the capsular polypeptide.

Colonies cultured on sheep blood agar are characteristically large and nonpigmented and have a dry “ground-glass” surface and
irregular edges. The colonies are quite sticky and adherent to the agar and, if the edge is lifted with a bacteriologic loop, it will
remain standing like beaten egg whites. Colonies are not hemolytic, in contrast with B. cereus. B. anthracis will appear
nonmotile in motility tests such as the microscopic observation of individual rods in a suspended drop of culture medium.

The definitive identification of nonmotile, nonhemolytic organisms resembling B. anthracis is made in a public health reference
laboratory. This is accomplished by demonstrating capsule production (by microscopy or DFA) and either specific lysis of the
bacteria with gamma phage or a positive DFA test for a specific B. anthracis cell wall polysaccharide. In addition, nucleic acid
amplification tests (e.g., polymerase chain reaction [PCR]) have been developed and are performed in reference laboratories.
Key Characteristics to Distinguish between B. anthracis & Other
Species of Bacillus

Characteristic Bacillus anthracis Other Bacillus spp.

Hemolysis Neg Pos
Motility Neg Pos (usually)
Gelatin hydrolysis Neg Pos
Salicin fermentation Neg Pos
Growth on PEA
blood agar Neg Pos
Bacillus cereus
 Diagnosis

Similar to B. anthracis, B. cereus and other species can be readily cultured from clinical specimens
collected from patients with the emetic form of food poisoning.

Because individuals can be transiently colonized with B. cereus, the implicated food (e.g., rice, meat,
vegetables) must be cultured for confirmation of the existence of foodborne disease.

In practice, neither cultures nor tests to detect the heat-stable or heat-labile enterotoxins are commonly
performed, so most cases of B. cereus gastroenteritis are diagnosed by epidemiologic and clinical criteria.

Bacillus organisms grow rapidly and are readily detected with the Gram stain and culture of specimens
collected from infected eyes, intravenous culture sites, and other locations.
TREATMENT, PREVENTION, AND CONTROL

Because the course of B. cereus gastroenteritis is short and uncomplicated, symptomatic treatment is
adequate.

The treatment of other Bacillus infections is complicated because they have a rapid and progressive course
and a high incidence of multidrug resistance (e.g., B. cereus carries genes for resistance to penicillins and
cephalosporins).

Vancomycin, clindamycin, ciprofloxacin, and gentamicin can be used to treat infections. Penicillins
and cephalosporins are ineffective. Eye infections must be treated rapidly. Rapid consumption of foods after
cooking and proper refrigeration of uneaten foods can prevent food poisoning.
Self-study material

• Murray last edition, chapter 20

Vibrio spp and Aeromonas
 Medically important bacteria

Flexible
Lacking cell walls Rigid cell walls
(spirochetes)
Mycoplasma Borrelia
Ureaplasma Leptospira
Treponema
Obligate intracellular
Filamentous Free-living
parasite

Actinomyces Chlamydia
Mycobacterium Rickettsia
Nocardia Coxiella
Gram positive Gram negative

Cocci (aerobic) Rods

Cocci Rods
Neisseria Aerobic Anaerobic
Aerobic Anaerobic Aerobic Anaerobic
Bacteroides
Fusobacterium
Staphylococcus Peptostreprococcus Corynebacterium Clostridium Enterobacteriaceae Others
Streptococcus Listeria Bacillus
Enterococcus Lactobacillus Propionibacterium Campylobacter Acinetobacter
Enterobacter Helicobacter Bordetella
Escherichia Brucella
Klebsiella
Fusobacterium
Prevotella Haemophilus
Salmonella Legionella
Shigella Proteus
Moraxella
Pseudomonas
Vibrio
Yersinia
Serratia
Phylum Proteobacteria Garrity et
al., 2005
Class Gammaproteobacteria Garrity et al., 2005
Order Vibrionales Garrity and Holt, 2001
Family Vibrionaceae Véron, 1965
Genus Vibrio
• The second major group of gram-negative, facultatively anaerobic, fermentative rods are the genera
Vibrio and Aeromonas.

• These organisms were at one time classified together in the family Vibrionaceae and were
separated from the Enterobacteriaceae on the basis of a positive oxidase reaction and the presence
of polar flagella.

• These organisms were also classified together because they are primarily found in water and are
able to cause gastrointestinal disease.

• However, deoxyribonucleic acid (DNA) sequencing has established that these genera are only
distantly related and belong in separate families: Vibrio and Aeromonas are now classified in the
families Vibrionaceae and Aeromonadaceae, respectively.

• The genus Vibrio is a gram negative, facultative anaerobic, curved rod, member of the family of
Vibrionaceae.

• The genus Aeromonas is a gram negative, facultative anaerobic, fermentative rod, member of the
family of Aeromonadaceae.
Of more than 150 described Vibrio spp. ~12 cause infections in humans. Human diseases caused by pathogenic bacteria of the
Vibrio genus can be divided in two major groups: cholera and non- cholera infections. V. cholerae is the aetiological agent of cholera,
a severe diarrhoeal illness usually caused by ingestion of contaminated food or water, although person- to-person transmission is
also possible. Of note, V. cholerae can also be found in fresh water. Non- cholera Vibrio spp., such as V. parahaemolyticus and V.
vulnificus, cause vibriosis, a group of infections with different clinical manifestations depending on the pathogen species, route of
infection and host susceptibility.
Pathogenic Vibrio spp. share several biological, clinical and environmental characteristics that set them apart as
important human pathogens.
These bacteria share interesting genomic structures, including two chromosomes, which are frequently shaped by
recombination and intense horizontal gene transfer events.

Plasmids, including those encoding antimicrobial resistance, are also commonly found in Vibrio species.
All Vibrio spp. grow in warm (typically ≥15 °C and below 40°C) sea water and brackish water, and Vibrio cholerae
can also be found in fresh water. All species of Vibrio require sodium chloride (NaCl) for growth. Vibrio spp. can
persist in a free living state, colonize fish and marine invertebrates or be associated with plankton, algae and
abiotic detritus. Vibrio spp. can also form biofilms on biotic and abiotic surfaces, an ability that has an essential role
in their environmental persistence.

Most vibrios have polar flagella (important for motility) and various pili that are important for virulence (e.g., toxin
coregulated pilus [TCP] in epidemic strains of V. cholera). The cell wall structure of vibrios is also important. All
strains possess lipopolysaccharides consisting of lipid A (endotoxin), core polysaccharide, and an O
polysaccharide side chain. The O polysaccharide is used to subdivide Vibrio species into serogroups.
There are more than 200 serogroups of V. cholerae plus multiple serogroups of V. vulnificus and V.
parahaemolyticus. V. cholerae O1 and O139 produce cholera toxin and are associated with epidemics of
cholera. Other strains of V. cholerae generally do not produce cholera toxin and do not cause epidemic
disease.
V.cholerae serogroup O1 is further subdivided into serotypes (Inaba, Ogawa, and Hikojima) and biotypes
(Classical and El Tor). Strains can shift between serotype Inaba and serotype Ogawa, with Hikojima a
transitional state in which both Inaba and Ogawa antigens are expressed.

The classical and El Tor biotypes differ in a variety of phenotypic traits; the classical biotype was responsible
for the first six cholera pandemics (which are considered concluded), whereas the ongoing seventh pandemic
is caused by the El Tor biotype, which in 1961 replaced the classical biotype
 V. cholera O1 and O139 has evolved as one of the most
Virulence factors successful pathogen in the history of mankind. To attain
the fitness for survival, the pathogen has acquired a
number of MGEs belongs to different classes such as
prophages (CTXΦ, VGJΦ, RS1, TLCΦ), pathogenicity
islands (VPI-1, VPI-2, VSP-1 & VSP-2) and integrative
conjugative elements (ICEs). The key virulence factor of
cholera, cholera toxin (CT) is encoded by
the ctxA and ctxB genes that induces the secretion of fluid
and electrolytes from the intestinal epithelial cells and
causes the diarrhea. CT is acquired through irreversible
integration of a single stranded DNA (+ssDNA) phage
CTXΦ into the dif sites of either or both the chromosome
of V. cholerae. The second most crucial virulence factor of
cholera pathogen, toxin-coregulated pilus (TCP), encoded
by the genes present in the TCP locus of VPI-1, helps the
pathogen in colonization in the gastrointestinal tract of the
host and also act as a cell surface receptor for CTXΦ.
This altogether suggests that the acquisition of the MGEs
is the key for the fitness and evolution of the cholera
pathogen for different pandemics.
Genetic organization of VPI-1 and integration mechanism
The cholera toxin is a complex A-B toxin that is structurally and functionally similar to the heat-labile enterotoxin of
enterotoxigenic Escherichia coli (ETEC). A ring of five identical B subunits of cholera toxin binds to the ganglioside
GM1 receptors on the intestinal epithelial cells. The active portion of the A subunit is internalized and interacts with G
proteins that control adenylate cyclase, leading to the catabolic conversion of adenosine triphosphate (ATP) to cyclic
adenosine monophosphate (cAMP). This results in a hypersecretion of water and electrolytes.
The means by which other Vibrio species cause disease is less clearly understood, although a variety of
potential
virulence factors have been identified. Most virulent strains of V. parahaemolyticus produce adhesins, a
thermostable direct hemolysin (TDH; also called Kanagawa hemolysin), and type III secretion systems that
mediate bacterial survival and expression of virulence factors. TDH is an enterotoxin that induces chloride ion
secretion in epithelial cells by increasing intracellular calcium.

An important method for classifying virulent strains of V. parahaemolyticus is detection of this hemolysin, which
produces -hemolytic colonies on agar media with human blood but not sheep blood. These virulent strains are
referred to as Kanagawa positive.

V. vulnificus and non-O1 V. cholerae produce acidic polysaccharide capsules that are important for disseminated
infections. V. cholerae O1 does not produce a capsule, so infections with this organism do not spread beyond the
confines of the intestine. In the presence of gastric acids, V. vulnificus rapidly degrades lysine, producing alkaline
by-products that neutralize the acids. In addition, the bacteria are able to evade the host immune response by
inducing macrophage apoptosis and to avoid phagocytosis by expression of a polysaccharide capsule. V.
vulnificus also possesses surface proteins that mediate attachment to host cells and secretes cytolytic toxins
leading to tissue necrosis.
Vibrio spp. differ in the routes of transmission to humans; non- cholera Vibrio spp., such as Vibrio
parahaemolyticus, Vibrio vulnificus and Vibrio alginolyticus, represent an important and growing source of
infections through contaminated seafood and direct exposure to water. The causative agent of cholera — V.
cholerae — is the only Vibrio sp. that has both human and environmental stages in its life cycle. V. cholerae can
be found in fresh water as free living, in clusters of many cells forming biofilms or in association with plankton (a
widely recognized environmental reservoir). From contaminated water, V. cholerae can be transmitted directly to
the human population. In contrast with other pathogenic non- cholera Vibrio spp., V. cholerae is also transmitted
person to person and through the faecal–oral route.
Prevention measures

Improvements in water supply, sanitation and food safety, coupled with community awareness, represent the
most effective means of preventing cholera and other diarrhoeal diseases.

Cholera vaccines have been very effective in controlling cholera; nevertheless, mass vaccination at the
country level is not always easy for several reasons, including political or cultural hesitancy in vaccine
acceptance, costs (as a booster dose of vaccine is also recommended) and lack of adequate supportive
infrastructure for the storage and delivery of vaccines.

Cholera vaccines. The first cholera vaccines developed required parenteral administration; however, this
administration route had limited efficacy and short duration of protection. As the oral route elicited increased
mucosal antibody responses against cholera, a shift from parenteral vaccines to oral vaccine development
occurred in the 1980s. Both live- attenuated and inactivated oral cholera vaccines (OCVs) have been
developed and tested.
Vibrio cholera vaccines
Vibrio spp treatment
 Laboratory diagnosis

Microscopy: Large numbers of organisms are typically present in the stools of patients at the onset of cholera, so
the direct microscopic examination of stool specimens can provide a rapid, presumptive diagnosis in cholera
outbreaks; however, as disease progresses the organisms are
diluted with massive fluid loss, and microscopy is less useful. Examination of Gram-stained wound specimens may
also be useful in a setting suggestive of V. vulnificus infection (e.g., exposure of susceptible individual to seafood or
seawater).

Immunoassays for the detection of cholera toxin or the O1 and O139 lipopolysaccharides are used for the
diagnosis of cholera in endemic areas. These tests have variable sensitivity
(as high as 97%) and specificity and have decreasing value as the disease progresses because fewer organisms
are present in the clinical specimens.

Nucleic Acid Amplification Tests are now widely used for diagnosis of enteric infections because they are rapid
and have high sensitivity compared with alternative tests. Most of these tests are multiplex tests, allowing detection
of multiple bacterial, viral, and parasitic enteric pathogens.

Culture Vibrios grow on most media used in clinical laboratories for stool and wound cultures, including blood agar
and MacConkey agar. Special selective agar for vibrios (e.g., thiosulfate citrate bile salts sucrose [TCBS] agar), as
well as an enrichment broth (e.g., alkaline peptone broth, pH 8.6), can also be used to recover vibrios in specimens
with a mixture of organisms (e.g., stools). Isolates are identified with selective biochemical tests and serotyped using
polyvalent antisera.
Aeromonas
• Aeromonas is a gram-negative, facultative anaerobic, fermentative rod that morphologically
resembles members of the family Enterobacteriaceae, now classificated as Aeromonadaceae.
• Almost 50 species and subspecies of Aeromonas have been described, many of which are
associated with human disease. The most important pathogens are A. hydrophila, A. caviae, and A.
veronii biovar sobria. The organisms are ubiquitous in fresh and brackish water.
• Aeromonas species cause three forms of disease: (1) diarrheal disease in otherwise healthy
people, (2) wound infections, and (3) opportunistic systemic disease in immunocompromised
patients (particularly those with hepatobiliary disease or an underlying malignancy).
• Although numerous potential virulence factors (e.g., endotoxin, hemolysins, heat-labile and heat-
stable enterotoxins) have been identified for Aeromonas, their precise role in disease is unknown.
• Acute diarrheal disease is self-limited, and only supportive care is indicated in affected patients.
Antimicrobial therapy is necessary in patients with chronic diarrheal disease, wound infections, or
systemic disease.
• Aeromonas species are resistant to penicillins, most cephalosporins, and erythromycin.
Fluoroquinolones are mostly active, even if resistance has been reported in strains recovered in
Asia.
Self-study material
• https://doi.org/10.1038/s41572-018-0005-8
• Murray last edition, chapter 26
• https://geoportal.ecdc.europa.eu/vibriomapviewer/
Secretory antibodies in the upper respiratory tract
Streptococcus
IgM pneumoniae
B lymphocytes (pneumococcus)

Dimeric
IgA
Polymeric Polysaccharide
J immunoglobulin capsule
chain receptor
(PigR)
Basolateral
Epithelial membrane
cell

Secretory
IgA
Secretory Antibody
opsonisation Streptococcus Apical
component pneumoniae membrane
Anti-capsular antibodies play an important role in defense against invading Streptococcus pneumonia species. The polysaccharide
capsule, however, is variable between strains with more than 90 serotypes currently identified. The variability of the capsule allows
a different bacterial strain to escape humoral responses generated against a previous infecting strain. In addition, the polysaccharide
capsule masks epitopes and also interferes with phagocyte receptors due to the negative charge. This also prevents bacteria from
entrapment in mucous. Colonisation of the upper respiratory tract is inhibited by IgM and IgA opsonising antibodies secreted across
the epithelium mediated by the polymeric immunoglobulin receptor (PigR).
Excretion of pathogens across upper respiratory tract epitheliium
IgM
B lymphocytes

Dimeric
IgA
Polymeric
immunoglobulin
J receptor
chain (PigR)
Basolateral
Epithelial membrane
cell

Pathogen
excretion Streptococcus Apical
pneumoniae membrane
Penetration of bacteria into the submucosa can be reversed by opsonisation with IgM or dimeric IgA secreted by B lymphocytes.
The polymeric immunoglobin receptor (PigR) binds the J chain of antibodies already bound to antigen and thereby facilitates the
excretion of pathogens by transcytosis.
Interference with IgA1 antibody efficacy

IgA1
cleavage
Fc
Streptococcus fragment
pneumoniae
Fab
fragment

IgA1

IgA1
protease

Streptococcus pneumoniae successfully colonises the nasopharynx in humans. The polysaccharide capsule varies between strains
and has a negative charge that resists entrapment in mucous. The polymeric immunoglobulin receptor (PigR) transports IgM and
dimeric IgA across the epithelium by transcytosis. These antibodies opsonise bacteria detected at mucosal surfaces and prevent
attachment. Bacteria detected in the submucosa can also be excreted by antibody-mediated transcytosis. The IgA1 isoform is
the most common type of secretory IgA. Streptococcus pneumoniae expresses a surface IgA1 protease that cleaves the Fc domain
from IgA1 which inhibits receptor-mediated phagocytosis. The Fab fragments remain bound and serve to mask other epitopes.
Complement evasion and inhibition of phagocytosis

C3b

C1

Complement
activation

Ply
Streptococcus
Host pneumoniae
cell lysis

LytA
Bacterial
antigens Bacterial
cell lysis

Phagocyte

Streptococcus pneumoniae has evolved a number of survival strategies such as interference with effective complement attack
and subsequent phagocytosis. In a self-sacrificing way some bacteria autolyse and divert immune attack away from living
bacteria. The LytA molecule expressed on the bacterial cell surface, once activated promotes lysis of the bacterium which
facilitates release of the intracellular toxin, pneumolysin (Ply). Ply activates complement and diverts the attack from other
bacteria. In higher concentrations, Ply can form a pore in the cell membrane of host cells and promote lysis. Release of bacterial
antigens excessively activate phagocytes via toll-like receptor engagement and allow bacteria to escape phagocytosis.
Complement evasion and inhibition of phagocytosis

Streptococcus
pneumoniae
Phagocyte
Factor B

PspC
Factor H
PspA Complement
receptor
C3b

Streptococcus pneumoniae also expresses cell surface molecules that disrupt complement protein binding. PspA prevents
C3b surface deposition. C3b usually initiates the alternative complement cascade and is also produced as an opsonin by the
classical and lectin complement cascades. Another surface protein, PspC, binds factor H, a negative regulator of the alternative
complement system, which prevents factor B binding to C3b to assemble the C3 convertase. Phagocytes expressing complement
receptors are thus inhibited from engulfing bacteria.
Escape from neutrophil extracellular nets

Antimicrobial
granules
DNA

Histone

Neutrophils

EndA

Streptococcus
pneumoniae

Neutrophils play an important role in innate immune control of bacteria, particularly Streptococcus pneumoniae. Neutrophils
classically employ phagocytosis and release of antimicrobial granules to control extracellular pathogens. However, a recently
discovered innate defense mechanism known as the neutrophil extracellular trap (NET) has been described. Chromatin (DNA
and histone proteins) with attached antimicrobial granules is extruded from the neutrophil into the environment and serves to
trap pathogens. The neutrophil dies during this process. However, Streptococcus pneumoniae evades capture by expressing
a DNA endonuclease, EndA, that cleaves the DNA and permits escape from the trap.