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Infectious Diseases and Translational Medicine
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Identification and Diagnosis of Newly
Emerging Pathogens
Yan Liu, Xuejie Yu
Abstract
From School of Basic Medical Sciences,
Department of Microbiology, Anhui
Medical University (Yan Liu);School
of Health Sciences, Wuhan University
(Xuejie Yu) .
Correspondence to: Xuejie Yu, Email:
[email protected].
Open access
DOI: 10.11979/idtm.201701005
Citation: Liu Y, Yu XJ. Identification and
Diagnosis of Newly Emerging Pathogens. Infect Dis Transl Med, 2017; 3(1):
12-16.
Copyright© The author(s) 2017. This
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data made available in this article, unless otherwise stated.
Received: Dec 1, 2016
Accepted: Jan 3, 2017
Published: Mar 14, 2017
Emerging infectious diseases (EIDs) are newly identified or known infectious diseases that
have either expanded in geographic range or increased in infection prevalence over the previous two decades. During the last three decades, more than 30 EIDs have surfaced worldwide,
including deadly diseases such as SARS, severe fever with thrombocytopenia syndrome
(SFTS), Ebola, Marburg virus disease, Nipah virus disease, hepatitis C, and AIDS.EIDs can
not only cause suffering and death in patients but also hinder commercial trade and travel, and
create fear or even widespread panic in society. The challenge for the medical community is to
effectively recognize and diagnose EIDs. The present study reports our experience in identification and diagnosis of EIDs.
Keywords: Emerging infectious diseases; Pathogen; Identification; Diagnosis
Introduction
C
lassical infectious diseases such as polio, smallpox, plagues, and measles are now
largely under control because of education, improved living conditions, enhanced
nutrition, and effective use of vaccines and antibiotics. However, despite remarkable advances in medical research and treatments in the 20th century, infectious diseases
remain one of the leading cause of death worldwide[1]. Most important infectious diseases
in the present day are emerging infectious diseases (EIDs), such as AIDS and Ebola. The
term, “emerging infectious disease” was coined in 1992 by the US National Academy of
Science’s Institute of Medicine (IOM) in its landmark publication, “Emerging Infections:
Microbial Threats to Health in the United States”. An EID could be a newly appeared
disease or a known disease whose incidence or geographic range is rapidly increasing,
or threatens to increase, in the near future within a population. The duration of emerging infections is defined as two decades[2]. A severe EID poses a serious potential threat
to public health because society is unprepared for diagnosis and control due to its lack of
knowledge and diagnostic methods. The present study reviews the factors affecting the
emergence of infectious diseases and strategies for diagnosis and prevention of EIDs.
1. Factors affecting EIDs
Many factors may affect the emergence of new infectious diseases or the re-emergence
of “old”/known infectious diseases (Table 1)[2].However, the most significant factors
for EIDs include: (1) genetic evolution of human pathogens; (2) cross-species spread of
pathogens from animals into the human population;(3) geographic range change of known
infectious diseases; and (4) emergence of multidrug-resistant bacterial infections. In the
first three, new pathogens will emerge in a population, and affect diagnosis, prevention,
and control of these emerging diseases due to unpreparedness for diagnosis. In the fourth
scenario, no new pathogen has emerged, though the infection can lead to inadequate or
delayed antimicrobial therapy, and is associated with worse patient outcomes. The focus
herein is on the first three scenarios with emergence of new pathogens.
1.1 Evolution of human pathogens
Genome assortment is the major reason many viruses can change in virulence. Reassortment is the mixing of the genetic segments of two multi-segmented RNA viruses into a
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Infect Dis Transl Med 2017;3(1):12-16.
Identification and Diagnosis of Newly Emerging Pathogens
Table1. Pathogens of emerging infectious diseases since 1970
Year of
discovery
Pathogen
Year of
discovery
Infection source
Pathogen
Infection
source
1973
Rotavirus
Human
1989
Hepatitis C
Human
1975
Parvovirus B-19
Human
1991
Guanarito virus
Mouse
1976
Cryptosporidium parvum
Fecal-oral
1991
Encephalitozoonhellem
Birds
1977
Ebola virus
Bat
1992
Vibrio cholerae O139
Aquatic
product
1977
Legionella pneumophila
Amoeba
1992
Bartonellahenselae
Cat
1977
Hantaan virus
Mouse
1993
Sin Nombre virus
Mouse
1977
Campylobacter jejuni
Cattle, Sheep, Dog, Poultry
1993
Encephalitozooncunculi
Rabbit
1980
HTLV-I
Human
1993
Anaplasmaphagocytophilum
Tick
1982
Escherichia coli O157:H7
Cattle, Sheep, Pig, Chicken
1994
Sabia virus
Rodents
1982
HTLV-II
Human
1995
HHV-8
Human
1982
Borreliaburgdorferi
Tick
1999
West Nile virus
Mosquito
1983
HIV/AIDS
Orangutan
1999
Nipah virus
Bat，Mouse
Helicobacter pylori
Human
2002
SARS Virus
Bat
1985
Enterocytozoonbieneusi
Pig, Human
2009
H1N1 Virus
Birds
1986
Cyclosporacayatanensis
Fecal-oral
2012
SFTS virus
Tick
1988
Hepatitis E virus
Human
2010
Chikungunya
Mosquito
1989
Ehrilichiachafeensis
Tick
2013
MERS-CoV
Camel
1984
new combination to form a new species of virus. The
genome of the influenza A virus consists of eight RNA
segments. This RNA is replicated in the nucleus of the
host cell. The new segments are transported to the cytoplasm and then assembled into new virus particles that
bud from the cell. When two virus strains infect a single
host cell, the RNA segments of both viruses may be assembled into a single virus at the plasma membrane. This
reasserted strain may possess properties of both parental strains. Moreover, domestic and wild animals such
as pigs, chickens, and ducks are natural influenza virus
hosts, serving as mixing vessels for creating reassortment
of the influenza virus. The reassortment may obtain the
ability to invade human cells, posing a potential disaster
for the human population, which is not immune to the
previously nonexistent virus. Reassortment is responsible
for some of the major influenza virus epidemics throughout history. The 1957 and 1968 pandemic flus were
caused by a reassortment of an avian virus and a human
virus[3]. Avian H5N1 influenza (or bird flu) causes death
in more than half of all cases, but it is limited in causing
human infection because it lacks efficient ability to pass
between humans, whereas the 2009 H1N1 influenza (or
swine flu virus) carried an unusual mix of swine, avian,
and human influenza genetic sequences, and transmitted
easily among humans. Fortunately, the H1N1 virus was
much less deadly than H5N1. Emergence of an influenza
virus as deadly as H5N1 and capable of spreading from
person to person could pose a global-scale threat for
public health. Deadly pathogens can travel at high speeds
around the world via airplane an passenger, which quickly spreads the pathogens across borders and overseas.
1.2 Cross-species spread of pathogens from animals
into human population
EIDs are new diseases in the human population, but their
pathogens may have already existed in their natural invertebrate or vertebrate animal hosts for million years.
When humans interact with the animal hosts, infectious
agents in animals are passed to the humans, causing
EIDs. These pathogens have co-evolved with their animal hosts for millions of years and are usually low or
non-pathogenic to these natural hosts. However, when
transmitted into a human population they may cause
deadly diseases. The frequency of zoonosis emerging in
a human population will rise as results of human popula13
Infectious Diseases and Translational Medicine
tion intrude into new geographical regions, such as what
occurs destruction of forested areas, or through increased
animal population due to farming or the increased natural
population of wild animals. Severe fever with thrombocytopenia syndrome (SFTS) is an emerging hemorrhagic
fever caused by the tick-borne bunyavirus SFTSV. SFTS
was first reported in China in 2011, while SFTSV may
have been circulating in central China for centuries before spreading rapidly to other parts of the country by
way of animals and ticks. In recent years the tick population has increased due to the rising populations of farm
animals.
1.3 Geographic range change of old/known infectious
diseases
An old/known infectious disease may change its geographic range and emerge in a new location. This is
mainly what occurred in the mosquito-borne virus genus
Flavivirus including West Nile virus (WNV), Zika virus,
and Chikungunya virus. These viruses were spread into
new areas by their vectors and through host animals. Flaviviruses are enveloped, single-stranded, positive-sense
RNA viruses around 10,000–11,000 bases with a diameter of 40–65 nm. Most such viruses are transmitted by
the bite of an infected arthropod (mosquito or tick) and
thus classified as arboviruses.
1.3.1 Spread of West Nile virus
WNV was first isolated from a febrile patient from the
West Nile district of Northern Uganda in 1937[4].Subsequently WNV epidemics were reported in the Mediterranean basin in the 1950s and 1960s in Israel and Egypt[5-7].
In 1996, the frequency, severity, and geographic range of
the outbreaks dramatically increased. Outbreaks of WNV
meningitis and encephalitis primarily affecting adults
were also reported that year in Bucharest, Romania[8].
This was the first urban WNV outbreak, and most symptomatic cases involved central nervous system infection[9], suggesting the virus’ changing epidemiology[10].
WNV crossed the Atlantic Ocean and reached the United
States in the summer of 1999 when a cluster of patients
with encephalitis was reported in the metropolitan New
York City area, and within 3 years the virus had spread
to most of the contiguous United States, and neighboring Canada and Mexico[11]. WNV has also been found in
Central and South America and in west China through
surveillance studies of field specimens, suggesting a potential risk for an outbreak in humans in these areas[12].
Since its discovery in the 1980s, WNV has spread across
a vast region of the globe and will continue to other areas
where its vector Culex mosquitoes exist.
1.3.2 Spread of Zika virus infection
Zika virus is transmitted by many Aedes spp. mosquitoes. It was identified in 1947 in rhesus monkeys during
sylvatic yellow fever surveillance in the Zika Forest in
Uganda, and was reported in humans in 1952[13].The virus circulated in forests among wild primates and arboreal mosquitoes, such as Aedes africanus[14]. Since the first
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description of Zika virus infection, the virus was thought
to only cause mild and sporadic human infections in Africa and Asia. The profile of Zika dramatically changed
during the large-scale outbreaks in Micronesia, French
Polynesia, and New Caledonia, respectively in 2007 and
2013–2014[15-17]. A new challenge has arisen in Zika’s
global spread. A widespread Zika epidemic was reported
in South and Central America and the Caribbean in
2015,with apparent increased incidence of microcephaly
in fetuses born to infected mothers posing a major concern[18]. As of July 13, 2016, the World Health Organization (WHO) had reported 65 countries and territories had
given evidence of Zika virus transmission since 2007,
including 48 with their first reported outbreak in or after
2015[19].
1.3.3 Spread of Chikungunya virus
Chikungunya is a viral disease that causes fever and severe joint pain. The Chikungunya virus was first isolated
after a 1952–1953 epidemic in Tanzania. The virus circulates in sub-Saharan forest regions of Africa, involving
nonhuman primate hosts and arboreal mosquito vectors
in ancestral transmission cycles [20-21]. Outbreaks have
been subsequently identified in Asia during the 1950s
and 1960s and were confined to tropical regions around
the Indian Ocean. Since 2004, the African lineage of the
virus has been spreading to several Indian Ocean islands
and to India, and subsequently in Europe, Asia, and
the Americas[22]. In the summer of 2014, the virus was
reported in the United States, and though its range has
been limited to the state of Florida, it likely will spread
to other states.
2. Control of EIDs
EIDs usually occur in a local population where the
pathogen is present or was recently imported. They may
repeatedly infect the local population until recognition
and notification by health care workers. An EID may be
misdiagnosed as a known infectious disease because of
nonspecific syndromes and a lack of diagnostic methods.
An unrecognized endemic EID may evolve into an epidemic or even pandemic disease if the agent is capable
of spreading readily from person-to-person and able
to sustain itself within the population, such as characteristic of severe acute respiratory syndrome (SARS).
SARS emerged in southern China in 2002 and was not
recognized as a new disease until it spread rapidly to
other parts of the country and later to other countries,
becoming a national and international pandemic. More
than 8,000 people had been infected and more than 800
died before a global effort finally halted the spread of
SARS[23]. Early diagnosis of EIDs and proper isolation
of patients may therefore succeed in controlling and preventing spread leading to epidemic or pandemic levels.
3. Strategy for identifying EID pathogens
An EID is a new disease in a population and physicians
Identification and Diagnosis of Newly Emerging Pathogens
have no knowledge of or experience with its diagnosis.
This may result in misdiagnosis and mishandling of patients, and may lead to interpersonal transmission. SFTS,
for instance, is very often mistreated and results in nosocomial transmission among family attendants in both
healthcare and community settings[24-25]. Nosocomial
transmission has been a major cause of morbidity and
mortality in Ebola endemic areas since the first outbreaks
described in the Democratic Republic of the Congo in
1976[26].
3.1 Sample collection
Acute blood sample within 2 weeks of disease onset
should be collected sterilely. Blood should be transported
on ice to a laboratory and processed immediately. If the
sample can not be processed immediately, it may be
stored at 4℃ for 1week or stored indefinitely at −80℃
before being accessed.
3.2 Pathogen isolation from EID patients
A majority of EIDs are caused by viruses or rickettsial
organisms, which are difficult to isolate. In the initial
diagnosis of an unknown infectious disease, a regular
bacterial culture should be performed to rule out bacterial infection. Simultaneously, the patient’s serum or anticoagulated blood sample should be processed in isolation
from viruses or rickettsia, respectively. Buffy coat or
white blood cells may be isolated from an anti-coagulated blood sample for cultivation of intracellular bacteria
such as Anaplasma, Ehrlichia, and Rickettsia. Cell infection is monitored by cytopathic effect, which is caused by
death or lysis of a virally or rickettsia-infected cell. CPE
include rounding of the infected cell, cell fusion to form
syncytia, and the appearance of inclusion bodies in nuclei or cytoplasm[27].Vero cells are most frequently used
to isolate viruses. However, many deadly human viruses
do not cause visible CPE in Vero cells, making it difficult
to judge whether the cells are infected. Therefore, other
cells may be used for isolation of viruses, and these may
develop CPE upon viral infection. For example, in initial
isolation of SFTSV both Vero cells and DH82 cells were
used[28]. In initial isolation, SFTSV does not form CPE
in Vero cells, but does in DH82 cells. A DH82 cell is a
monocyte cell line with a round form that loosely attaches to the plate surface, but it will mature into a fibroblastlike macrophage that tightly attaches to the plate upon
infection by a virus or by intracellular bacteria.
3.3 Molecular identification of pathogens of EIDs
Isolation of an EID’s pathogen takes several days, or
even weeks, which delays diagnosis and treatment.
Molecular identification of EID pathogens allows rapid
detection and identification of organisms. The pathogen should be identified in the early stage of diagnosis
and after being isolated. Before performing molecular
identification, the pathogen must be roughly determined
based on seasonal epidemiology, the patient’s geographic
origins, and clinical symptoms. For instance, if the patient comes from tropical endemic areas in Africa, Asia,
or South America, then Zika, WNV, Chikungunya, yellow fever, dengue, and Ebola should be considered. A
patient with no traffic history before onset of the disease
should be questioned regarding history of contact with
animals and arthropod bites to determine whether there
is infection with an arthropod- or animal-borne pathogen. Molecular diagnosis involving polymerase chain
reaction (PCR) should include a broad range of PCR
primers targeting several pathogens in a single genus or
family of potential pathogens to amplify the unknown
pathogens. In identifying a new virus, the virus may have
no sequence homology with any known virus. In such a
case, the new virus can be sequenced by next-generation
sequencing (NGS), which can produce an enormous volume of sequences, with high speed and throughput. NGS
techniques have been applied to metagenomics-based
strategies for detection of unexpected disease-associated
viruses and for discovery of novel human viruses[29].
4. Principles for determining the pathogen as the
cause of a novel infectious disease
Virus or bacterium associated with patients are not necessarily the pathogen and it may be only an accompanying
organism[30], for example, Chlamydia was discovered in
SARS patients. The virus or bacterium from the patient
needs to be tested to confirm it is the EID’s pathogen.
Koch’s postulates are used to determine the causative
agent of a particular disease. The postulates have previously been used guide identification of pathogens. However, in Koch’s time, there was no virology or molecular
biology; therefore, Koch’s postulates have limitations in
the molecular biology and metagenomic era. First, many
fastidious growth pathogens are difficult to cultivate,
and detection of a pathogen’s genetic material by reverse
transcription PCR or standard PCR may be used as evidence for infection. Second, many pathogens have no
susceptible animal model, which renders it impossible to
deny the pathogen caused human disease.
5. Summary
EIDs are either new diseases or known diseases that,
over the previous two decades, have expanded in geographic range or have a significantly increased incidence
rate. New infectious diseases and reemerging infectious
diseases both pose a challenge for physicians and public
health workers because of the novelty of the disease in a
certain population and the lack of knowledge of the disease and of prepared diagnostic methodology. Delayed
diagnosis or misdiagnosis of an EID may cause great
fear in populations. An unrecognized endemic EID may
evolve into an epidemic, or even pandemic, disease if
the agent has the ability to readily spread from person to
person and to sustain itself within the population, such as
was seen with SARS. Therefore, early diagnosis of EIDs
is important for the health in the local and global populations. The strategy for diagnosing EIDs includes: (1)
15
Infectious Diseases and Translational Medicine
recognition of EID by clinical symptoms; (2)isolating,
and identifying the pathogen from, the patients; (3)determining whether the isolated organism is the pathogen
that causes infection. Because EIDs may be contagious,
patients should be isolated before diagnosis and until
recovery. In the era of easily accessible airplane travel,
infectious diseases have no boundaries and spread at jet
speed, which requires the world to corporate in diagnosing and containing EIDs.
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