MEDICAL DIAGNOSTIC LABORATORIES, L.L.C.

Respiratory Tract Infections

Sinusitis Human Metapneumovirus

Streptococcus pneumoniae,
Common Cold - Rhinoviruses, Haemophilus influenzae
Coronaviruses, Adenoviruses, Myxoviruses,
Coxsackie Viruses A and B, Mycoplasma pneumoniae,
Chlamydophila pneumoniae
Respiratory Syncytial Viruses A & B

Whooping Cough
Bordetella parapertussis,
Bordetella pertussis,
Bordetella bronchiseptica,
Mycoplasma pneumoniae,
Chlamydia trachomatis
Pharyngitis - Adenovirus, Bordetella pertussis
Bordetella parapertussis
Herpes Simplex Virus,
Coxsackie Viruses A and B,
Streptococcus pyogenes (GAS),
Corynebacterium diphtheriae

Group A Streptococcus

Influenza Viruses A & B

Bronchitis - Parainfluenza Viruses,

Respiratory Syncytial Virus,
Influenza Viruses, Mycoplasma pneumoniae,
Chlamydophila pneumoniae Human Parainfluenza Viruses 1-4
Bronchiolitis
Respiratory Syncytial Virus, Human
Metapneumovirus, Adenovirus, Parainfluenza
Viruses, Mycoplasma pneumoniae, Influenza Virus
MEDICAL DIAGNOSTIC LABORATORIES, L.L.C.

Respiratory Tract Infection (RTI)

The respiratory tract is

C o st (B illio n s US $)
susceptible to infection by
0 20 40 60 80 100 120 140
a host of bacterial and viral
pathogens; the fact that C a nce r 116. 9
they share similar clinical Dia be te s M e llitu s 114. 6
presentations makes C HD 100. 8
differentiation and diagnosis A rth ritis 82. 5
difficult. In response to S tro k e 45. 4
this, Medical Diagnostic Hy pe rte n sio n 40. 4
Laboratories, L.L.C. (MDL) has designed a series of V ira l R T I 39. 5
tests for the determination of respiratory pathogens COP D 30. 4
utilizing the NasoSwab™ collection and transport C HF 21
device. Expanding upon its OneSwab® and UroSwab® 17. 7
O ste o pe ro sis
methods of patient sampling, MDL’s NasoSwab™
A sth m a 14. 3
represents a less invasive method of sample
M igra in e 14. 2
procurement that utilizes Flocked Swab Technology.
O titis M e dia 6. 1
A lle rgic R h in itis 1. 6
Table 1. The NasoSwab™ collection method was directly
compared to nasal lavage in a blinded study comprised of 98
pediatric patients. Seven Real-Time PCR assays developed by Figure 1. Economic impact of viral respiratory tract infections in
MDL were used in the comparison. comparison to other common medical disorders. Adapted from
(3).
Pathogen Lavage (%) NasoSwab™ (%)
Human
13 (13.3) 12 (12.2) The 2007 National Vital Statistics Report (4) lists
metapneumovirus
RSV A 15 (15.3) 18 (18.4) chronic lower respiratory diseases and influenza/
RSV B 0 (0.0) 1 (1.0) pneumonia as the fourth and seventh leading
Influenza A 2 (2.0) 2 (2.0) causes of death, respectively, when considering
Influenza B 2 (2.0) 6 (6.0) all ages. When analyzed by individual age group,
B. parapertussis 0 (0.0) 0 (0.0) chronic lower respiratory diseases are within
B. pertussis 0 (0.0) 0 (0.0) the ten leading causes of death for ages 19 and
under and 45 and up, while influenza/pneumonia
Determination, when performed, is typically via the remains a risk factor for all age groups (4, 5). Such
somewhat invasive and uncomfortable method of statistical studies help define the at-risk populations
nasal lavage, which typically serves as a deterrent as infants 6 months of age and younger, as well
for most patients. The search for a less invasive as the elderly and adults already diagnosed with
method of sampling led to the development of a respiratory tract condition, such as Chronic
several generations of nasal swabs, differing in Obstructive Pulmonary Disease (COPD) or asthma
their choice of fibers (1). The clinical utility of these (5, 6). Within these populations, the burden of RTI
precursors was not proven, abilities and diagnostic is greater due to the lack or compromise of a fully
values were consistently out-performed by nasal developed immune system.
lavage (1). Some swab variations, particularly
calcium alginate, were found to inhibit downstream From an economic standpoint, lower respiratory
PCR analyses (2). The flocked nature and conical tract infections were the seventh most costly
shape of the MDL NasoSwab™ addresses this malady in 2003, with 39.5 billion dollars spent
issue. A blinded comparison of NasoSwab™ on treatment (Figure 1) (3). This figure is further
to nasal lavage collection methods revealed broken down into several factors to show the
substantial agreement between the two methods absolute cost of respiratory illness to an individual
following Real-time PCR analyses (Table 1). family, taking into consideration variables like
www.mdlab.com • 877 269 0090
missed work time along with physician and
treatment costs (Figure 2) (3). A separate study
performed by a community teaching hospital
in Illinois reported that the ability to identify
the causative agent, whether bacterial or viral,
results in significant decreases in the length of
patient hospitalizations, patient mortality and
antibiotic dispensation (8). When clinical testing
included identification of the viral respiratory
agent, expenditures were significantly reduced;
the average length of a hospital stay was halved,
dropping from 10.6 days the previous year to 5.3
in the following year, representing an average
savings of almost $6,000 per patient (8).
Figure 2. Economic impact of viral respiratory tract infections analyzed
by individual variables. Adapted from (3).
Considering the physical and economic burdens
RTIs present for every age group, identification of
the causative agent will allow for more appropriate
treatment of patients and at the same time may
reduce the costs of healthcare. MDL offers the
NasoSwab™ as a means of rapid identification
of respiratory tract infections to enable accurate
diagnosis and subsequent patient management.
NasoSwab™ is supplied in two sizes, adult and
infant, for broader applicability and use within
populations of different ages. The infant swab was
designed with a guard along its shaft regulated to
the depth of the infant’s nasal passages to ensure
proper depth is achieved during the swabbing
process. Real-Time PCR assays were developed
for the identification of viral and bacterial pathogens
capable of infecting the respiratory tract.
Sinusitis
Streptococcus pneumoniae,
Common Cold - Rhinoviruses,
Coronaviruses, Adenoviruses, Myxoviruses,
Haemophilus influenzae
Coxsackie Viruses A and B, Mycoplasma pneumoniae,
Chlamydophila pneumoniae
Whooping Cough
Bordetella parapertussis,
Bordetella pertussis,
Bordetella bronchiseptica,
Mycoplasma pneumoniae,
Chlamydia trachomatis
Pharyngitis - Adenovirus,
Herpes Simplex Virus,
Coxsackie Viruses A and B,
Streptococcus pyogenes (GAS),
Corynebacterium diphtheriae
Bronchitis - Parainfluenza Viruses,
Respiratory Syncytial Virus,
Influenza Viruses, Mycoplasma pneumoniae,
Chlamydophila pneumoniae
Bronchiolitis
Respiratory Syncytial Virus, Human
Metapneumovirus, Adenovirus, Parainfluenza
Viruses, Mycoplasma pneumoniae, Influenza Virus
Figure 3. Common respiratory tract manifestations and their
associated pathogens.
References:
1. Stensballe LG, Trautner S, Kofoed PE, Nante
E, Hedegaard K, Jensen IP, Aaby P. 2002.
Comparison of nasopharyngeal aspirate and
nasal swab specimens for detection of respiratory
syncytial virus in different settings in a developing
country. Trop Med Int Health. 7:317-21.
2. Cloud JL, Hymas W, Carroll KC. 2002. Impact
of nasopharyngeal swab types on detection of
Bordetella pertussis by PCR and culture. J Clin
Microbiol. 40:3838-40.
3. Fendrick AM, Monto AS, Nightengale B, Sarnes
M. 2003. The economic burden of non-influenza-
related viral respiratory tract infection in the United
States. Arch Intern Med. 163:487-94.
4. National Vital Statistics Report. 2007. Health, US.
55.
5. Winter JH. 1991. The scope of lower respiratory
tract infection. Infect 19 Suppl 7:S359-64.
6. Greenberg SB. 2002. Viral respiratory infections in
elderly patients and patients with chronic obstructive
pulmonary disease. Am J Med. 112 Suppl 6A:28S-
32S.
7. Brixner DI. 2004. Clinical and economic outcomes
in the treatment of lower respiratory tract infections.
The Am J Man Care. 10:S400-7.
8. Barenfanger J, Drake C, Leon N, Mueller T,
Troutt T. 2000. Clinical and financial benefits of
rapid detection of respiratory viruses: an outcomes
study. J Clin Microbiol. 38:2824-8.
MEDICAL DIAGNOSTIC LABORATORIES, L.L.C.
Bordetella pertussis & Bordetella
parapertussis
Pertussis, commonly known as
whooping cough, results from an
upper respiratory tract infection with
Bordetella pertussis or Bordetella
parapertussis. A highly contagious
and endemic disease, cases
induced by infection with Bordetella
parapertussis have been found to occur less frequently than
those resulting from B. pertussis infection and often result in
less severe infections (15). This infection was a significant
cause of childhood morbidity and mortality in the early part of
the twentieth century and reemerged as a significant health
concern in the late 1990’s. The lowest point in the incidence
of pertussis infection was attributed to the development and
widespread use of pertussis vaccines. In 2002, 8,296 cases
of pertussis were reported for an incidence of 3.01 cases per
100,000 people (5). Although a significant decrease from the
baseline annual morbidity in 1922 (147,271 cases), the case
than that reported in 2002 is a greater than 49% increase in
pertussis cases reported in the 1980’s (6, 16). The upward
trend in childhood pertussis cases has been primarily
attributed to decreased vaccine usage, while the increase
in adult pertussis cases is contributed to waning vaccine-
induced immunity and the lack of a vaccine approved for
use in adults (16). A third possibility, whereby the bacterium
has genetically adapted itself to circumvent the vaccine, has
also been demonstrated by an almost fifty year retrospective
epidemiological study conducted in the Netherlands (15). This
study involved the genetic fingerprinting of archived pertussis
strains using seven year intervals for the comparative study
(Figure 1). An initial decrease in genetic diversity was
observed following the institution of widespread vaccination,
followed by a period of increased genetic diversity. Genetic
sequencing analyses revealed mutations within two key
virulent factors, pertussis toxin and pertactin, to be associated
with these expanding, diverse strains and the ability to
circumvent vaccination strategies (15). Pertussis is the
least well controlled of the reportable, vaccine preventable
childhood diseases in the United States for which universal
vaccination has been recommended (14).
Vaccination Strategies and Schedules
Factors that influence the susceptibility to pertussis infection
include age, early administration of antibiotics before the
onset of cough and immunity conferred by vaccination (14).
A greater importance is given to the role of immunization
since younger children are more susceptible to infection
due to their lack of mature immune systems and antibiotic
intervention following onset of cough has been deemed of
limited efficacy. Unfortunately, the protection conferred by
childhood pertussis vaccination wanes five to ten years post
completion of childhood vaccination schedules, thereby
leaving adolescents and adults susceptible (14). In 2005,
the United States licensed Tdap, an acellular vaccine that
confers protection against tetanus, diphtheria and pertussis
for use in adolescents and adults ages eleven to sixty-four
(14). The Advisory Committee on Immunization Practices
recommends the following Tdap administration schedule:
a single dose to individuals between the ages of nineteen
and sixty-four as either a booster or initial vaccination
administration, administration as a booster every ten years,
administration as a preventive measure for any individuals
who are or will be in immediate contact with children under
the age of twelve months and all health care workers having
direct contact with patients (14).
Figure 1: Increased genetic diversity of Bordetella pertussis
following the initiation of widespread vaccination. Adapted from (15).
Pathology
B. pertussis is a fastidious gram-negative coccobacillus
that enters the respiratory tract through the inhalation of
contaminated aerosols. The organism releases toxins that
damage the airway epithelium to aid in host entry. While
whooping cough can be caused by other Bordetella species,
B. parapertussis, B. bronchiseptica and B. holmseii, B.
pertussis remains the leading pathogenic cause. Illness
caused by these other species is not preventable by
vaccination (14). Colonization is achieved by the preferential
adherence of B. pertussis to ciliated epithelium in the upper
respiratory tract. This interaction is primarily mediated by
filamentous hemaglutin (FHA) and Pertussis Toxin (PT) (3, 4,
11, 12, 13). FHA is also involved in the subsequent invasion
of respiratory epithelial cells (11, 12). PT disrupts phagocytic
functions reducing the body’s ability to clear an infection
(1). Reduced mucous movement and coughing results
from decreased ciliary beat frequency mediated by the B.
pertussis tracheal cytotoxin (7, 9). The typical incubation
period ranges from seven to ten days. The catarrhal and
early paroxysmal stages of infection, which can last as long
as six weeks, are the most infectious time frames and are
often indistinguishable from other early stage respiratory
infections (14).
Symptoms
Infection with B. pertussis is characterized by three phases:
the catarrhal, paroxysmal and convalescent phases. The
catarrhal phase is generally characterized by fever, coughing,
nasal congestion and malaise and lasts approximately ten
days. These symptoms are indistinguishable from other upper
respiratory infections. The paroxysmal phase is characterized
www.mdlab.com • 877 269 0090
by prolonged coughing followed by a characteristic “whoop”
or inhalation as the patient attempts to catch his/her breath.
In older children and adults, characteristic phases may not
be observed. Instead these individuals typically present with
prolonged coughing and headaches. Major complications,
including hypoxia, apnea, pneumonia, seizures, and
encephalopathy. Malnutrition can also occur, most often in
infants or young children. In 2003, thirteen children died from
complications of pertussis (5).
Transmission
B. pertussis is typically transmitted through direct contact with
respiratory secretions and/or aerosols. It is not uncommon
for colonized, older children to inadvertently expose their
younger, unvaccinated siblings to B. pertussis. Patients are
typically highly contagious during the catarrhal phase of
infection, but are also able to transmit bacteria up to three
weeks after the onset of coughing. Up to 90% of susceptible
household members develop pertussis after exposure to an
infected individual (5).
Diagnosis
The World Health Organization (WHO) has defined pertussis
as a paroxysmal cough present for greater than 21 days in
combination with either culture of B. pertussis or positive
serology or contact with a culture-positive individual (17).
Culture of B. pertussis can be hampered by the time point
during the course of infection at which a sample is taken and
by the age of the patient (10). Often B. pertussis cannot be
cultured from patients that have entered the paroxysmal phase
or from adult patients. Polymerase Chain Reaction (PCR) has
also been used to diagnose B. pertussis infections. However,
PCR, like culture, is more likely to be positive in the early stages
of disease and less sensitive in adults (8). Current serology for
B. pertussis infection is largely based upon antibody reactivity
to FHA and PT. The presence of IgA is indicative of a natural,
recent infection, whereas IgG appears during the paroxysmal
phase and remains detectable for more than six months.
MDL utilizes the ViraStripe system for the detection of B.
pertussis-specific IgG and IgA antibodies in serum. This
Western blot based system detects antibody reactivity to
filamentous hemagglutinin (220 kD) and the B subunit of
Pertussis toxin (28 kD). The B. pertussis IgG ViraStripe has
a sensitivity of 100% and a specificity of 97 to 98%. The B.
pertussis IgA ViraStripe has a sensitivity of 100% and a
specificity of 96 to 100%.
Pertussis serology may not be of clinical diagnostic value
for infants less than three months of age due to the under
development of the infants’ IgA response mechanism at
this early age or in cases involving IgA-deficient patients of
any age. For this reason, as well as for the early detection
of respiratory diseases, MDL has expanded its testing to
include two Real-Time PCR-based assays to be used in
conjunction with their latest method of respiratory sampling,
the NasoSwab™, for the detection of both B. pertussis and B.
parapertussis. Previous studies have demonstrated the utility
of both Dacron and Rayon nasopharyngeal swabs for the
detection of pertussis species in PCR-based methodologies
and discounted the utility of calcium alginate-based swabs due
to their low sensitivity and specificity (2). NasoSwab™, due
to its flocked, nylon nature and unique flexible, tapered head
represents a marked advancement in respiratory swabbing
technology that mirrors both the sensitivity and specificity of
nasal lavage sampling methods in an easier, less invasive
format.
References:
1. Brown EJ, Newell AM, Gresham HD. 1987. Molecular
recognition of phagocytic function. Evidence for involvement of
a guanosine triphosphate-binding protein in opsonin-mediated
phagocytosis by monocytes. J Immunol. 139:3777-3782.
2. Cloud JL, Hymas W, Carroll KC. 2002. Impact of
nasopharyngeal swab types on detection of Bordetella pertussis
by PCR and culture. J Clin Micro. 40(10):3838-3840.
3. Carbonetti NH, Artamonova GV, Andreasen C, Dudley E,
Mays RM, Worthington ZE. 2004. Suppression of serum
antibody responses by pertussis toxin after respiratory tract
colonization by Bordetella pertussis and identification of an
immunodominant lipoprotein. Infect Immun. 72:3350-3358.
4. Carbonetti NH, Artamonova GV, Mays RM, Worthington
ZE. 2003. Pertussis toxin plays an early role in respiratory tract
colonization by Bordetella pertussis. Infect Immun. 71:6358-
6366.
5. Centers for Disease Control and Prevention. 2003. Pertussis.
www.cdc.gov/ncidod/dbmd/diseaseinfo/pertussis_t.htm
6. Centers for Disease Control and Prevention, 1999. Achievements
in Public Health, 1900-1999: Impact of Vaccines Universally
Recommended for Children – United States, 1990-1998.
48;243-248.
7. Cookson BT, Cho HL, Herwaldt LA, Goldman WE. 1989.
Biological activities and chemical composition of purified tracheal
cytotoxin of Bordetella pertussis. Infect Immun. 57:2223-2229.
8. Dragsted DM, Dohn B, Madsen J, Jensen JS. 2004.
Comparison of culture and PCR for detection of Bordetella
pertussis and Bordetella parapertussis under routine laboratory
conditions. J Med Microbiol. 53:749-754.
9. Goldman WE, Klapper DG, Baseman JB. 1982. Detection,
isolation, and analysis of a released Bordetella pertussis product
toxic to cultured tracheal cells. Infect. Immun. 36:782-794.
10. Granstrom, G, Wretlind B, Granstrom M. 1991. Diagnostic
value of clinical and bacteriological findings in pertussis. J Infect.
22:17-26.
11. Ishibashi Y, Nishikawa A. 2002. Bordetella pertussis infection
of human respiratory epithelial cells up-regulates intercellular
adhesion molecule-1 expression: role of filamentous
hemagglutinin and pertussis toxin. Microb Pathog. 33:115-125.
12. Ishibashi Y, Relman DA, Nishikawa A. 2001. Invasion of human
respiratory epithelial cells by Bordetella pertussis: possible role
for a filamentous hemagglutinin Arg-Gly-Asp sequence and
α5β1 integrin. Microb Pathog. 30:279-288.
13. Ishibashi Y, Claus S, Relman DA. 1994. Bordetella pertussis
filamentous hemagglutinin interacts with a leukocyte signal
transduction complex and stimulates bacterial adherence to
monocyte CR3 (CD11b/CD18). J Exp Med. 180:1225-1233.
14. Kretsinger K, Broder KR, Cortese MM, Joyce MP, Ortega-
Sanchez I, Lee GM, Tiwari T, Cohn AC, Slade BA, Iskander
JK, Mijalski CM, Brown KH, Murphy TV. 2006. Preventing
tetanus, diphtheria, and pertussis among adults: use of tetanus
toxoid, reduced diphtheria toxoid and acellular pertussis
vaccine. Centers for Disease Control and Prevention MMWR
Recommendations and Reports December 15, 2006/55(RR17);1-
33. http://www.cdc. gov/mmwr/preview/mmwrhtml/rr5517a1.htm
Accessed May 17, 2007.
15. Mooi FR, van Loo IHM, King AJ. 2001. Adaptation of Bordetella
pertussis to vaccination: a cause for its reemergence? Emerg
Infect Dis 7(S3):526-528.
16. Tanaka M, Vitek CR, Pascual FP, Bisgard KM, Tate JE,
Murphy TV. 2003. Trends in pertussis among infants in the
United States, 1980-1999. JAMA. 290:2968-2975.
17. World Health Organization. 1991. WHO meeting on case
definitions of pertussis, 10 to 11 January 1991, MIN/EPI/
PERT91.1, p.4-5. World Health Organization, Geneva,
Switzerland.
MEDICAL DIAGNOSTIC LABORATORIES, L.L.C.
Group A Streptococcus
Group A Streptococcus (GAS),
also referred to as beta hemolytic
Streptococcus pyogenes, is a
common pathogen of the upper
respiratory tract that is commonly
associated with acute bacterial
pharyngitis (strep throat), as well as
the cutaneous infections impetigo
and necrotizing fasciitis. Long-term, non-suppurative
complications associated with untreated or recurrent
infections include rheumatic fever, scarlet fever and
acute glomerulonephritis (2, 5). More recently, ever
increasing pathogenic strains have been emerging
which predominately affect the skin and soft tissue
and can lead to potentially life-threatening systemic
infections (4).
Pathology
Most streptococcal strains have a predilection for
the respiratory tract and GAS represents the most
common cause of bacterial pharyngitis (5). This genus
of bacterium represents a large and diverse group of
gram-positive, non-motile cocci that morphologically
tend to grow in pairs as short chains within clinical
specimens (4, 6). There are more than twenty known
streptococcus serotypes, designated alphabetically, of
which serotypes A and B are the most clinically relevant
(4). Streptococcus pyogenes, also referred to as Group
A Streptococcus, is a common human pathogen and
is estimated to be present asymptomatically in 5% to
15% of the population (6). Despite this asymptomatic
carriage, GAS is considered to be an opportunistic
pathogen such that the bacteria can become pathogenic
within these carriers when the host’s immune defenses
are compromised (6).
Symptomology
The incubation period for strep throat is 2 to 4 days
post-exposure. Once infected, individuals can
transmit infection for up to three weeks if they remain
asymptomatic; however, initiation of an antibiotic
regimen usually limits this time to approximately
twenty-four hours (2, 5). Despite the quick decline in
bacterial transmissibility, it is important that patients
complete the full course of antibiotic therapy in order
to prevent possible non-suppurative infections, the
most notable of which is acute rheumatic fever, from
occurring in the subsequent weeks following infection
(5).
Transmission occurs via direct contact with
contaminated saliva, aerosolized nasal discharge
or skin wounds of an infected individual with the
mucous membranes or via inhalation by non-infected
individuals. Crowded living conditions, such as
dormitories and nursing homes, have been shown to
increase the rate of transmission. Reports of tainted
milk and milk-based products serving as vehicles of
transmission have also been reported (2). Transmission
from asymptomatic individuals who serve as carriers
occurs in very rare instances.
Frequency
GAS infections are endemic worldwide. Within the
United States, upper respiratory tract infections (UTI)
predominate within northern regions and typically
follow a winter/spring seasonal pattern. UTIs rarely
affect neonates due to the transplacental exchange
of protective antibodies from mother to child but are
a common cause of pharyngitis among children older
than three years of age (4). The CDC reports that over
ten million cases of noninvasive GAS occur annually
(1). In contrast, streptococcus-induced skin infections
appear to predominate in warmer climates and can
occur year round.
Complications
Prior to the age of antibiotic therapy, S. pyogenes
infections posed a significant public health risk. Both
suppurative and non-suppurative complications can
occur following GAS infections. Suppurative infections
include otitis media and sinusitis within the respiratory
tract as well as streptococcal bacteremia, the cutaneous
infection, necrotizing fasciitis, and, rarely, meningitis or
brain abscess (5). Non-suppurative infections, including
rheumatic fever, post-streptococcal glomerulonephritis,
Sydenham’s chorea and streptococcal toxic shock
syndrome (sTSS), can occur weeks or months
following infection. Rheumatic fever, characterized by
joint and valvular heart disease, typically occurs within
three weeks of infection and was found to be the most
frequently occurring complication (1).
Vaccine
Currently, there is no vaccine available for GAS but
several companies are researching and developing
such a product. The impetus behind the development
of such a vaccine stems from the non-suppurative
infections GAS induces, most notably rheumatic fever.
www.mdlab.com • 877 269 0090
Treatment
Both the American Heart Association and the Infectious
Disease Society of America have chosen penicillin
as the treatment of choice for GAS infections despite
studies that have demonstrated higher clinical cure
rates to be associated with cephalosporin (5). Treatment
typically consists of a ten day course of either penicillin
or its derivative, amoxicillin; this single course is quite
effective at eradicating the bacterium so long as the
patient is fully compliant (5). Although rare, there have
been instances where the initial treatment failed and
required follow-up administration with a cephalopsporin
class drug. The ability to differentiate between true
infection and carrier status is critical.
Diagnosis
Due to the fact that both pharyngitis and tonsillitis
are induced by a number of respiratory pathogens,
a confirmatory diagnosis is recommended prior to
initiating treatment. Throat culture and point-of-care
tests are the most common methods while Polymerase
Chain Reaction (PCR)-based amplification methods are
on the rise.
Throat Cultures remain the most common confirmatory
method of diagnosis. This method requires that a swab
of the patient’s throat be streaked across a blood agar
plate, which promotes GAS growth, in the presence of
a bacitracin-impregnated disc. The use of blood agar is
confirmatory only for the presence of GAS in this assay,
whereby, the beta hemolytic activity associated with GAS
is evaluated based on the cultured bacterium’s ability
to lyse red blood cells and their sensitivity to the drug
bacitracin (4). Plates are incubated for at least twenty-
four hours, at which point they are monitored for growth.
Negatively scored plates are traditionally incubated for
an additional twenty-four hours for true confirmation.
The limitations of this method include the reporting of a
significant number of false negatives and the selective
evaluation for GAS only. This method has an estimated
10% false negative reporting rate associated with it; the
issue of asymptomatic GAS carriers, or false positives,
is resolved based on the lack of overt symptoms that
would confirm the presence of an active infection (5).
Also, the selective nature of the culturing procedure
neither allows for the evaluation nor identification of
other possible causative bacterium (5).
Point-of-care tests represent a significant increase
over traditional, culture-based methods having the
capability to identify causative pathogens in as few
as twenty minutes in some instances, allowing for
confirmatory diagnosis and treatment while the
patient waits. These assays are centered around the
identification of a bacterial-specific protein based on
its ability to bind a DNA probe in either an enzymatic-
based or a chemiluminescent–based assay (5). While
these tests are typically highly specific, having 95% or
greater specificities, they suffer from a lack of sensitivity,
which usually necessitates throat culturing as a follow-
up, confirmatory test for all negative specimens.
Real-Time PCR reactions, when designed and
validated properly, can be a fast and efficient means
of diagnosing infections. This method involves the
repeated amplification of specific genetic elements.
They also have added benefit of being able to speciate
among related pathogens.
MDL has developed a new method of respiratory tract
sampling, the NasoSwab™, and a series of Real-Time
PCR assays to aid physicians in discerning the causative
agents responsible for these overlapping symptoms.
The novel “flocked-swab” technology employed by
the NasoSwab™ represents a less invasive manner
of sampling the upper respiratory tract whose particle
retention, as measured by both level of specificity and
sensitivity by an independent, blinded study, matches
or exceeds the levels obtained with nasal washes. The
employ of specialized media allows for room temperature
storage of specimens which serves to preserve the
integrity of the pathogen better than does the immediate
freezing of nasal wash specimens.
Table 1. Probability of various etiological causes of
pharyngitis. Adapted from (7).
Etiology Probability, %
Viral 50-80
Streptococcal 5-36
Epstein-Barr virus 1-10
Chlamydia pneumoniae 2-5
Mycoplasma pneumoniae 2-5
Neisseria gonorrhoeae 1-2
Haemophilus influenzae type b 1-2
Candidiasis <1
Diphtheria <1
References:
1. Centers for Disease Control and Prevention: Division of
Bacterial and Mycotic Diseases. October 11, 2005, posting
date. Group A Streptococcal (GAS) Disease. Accessed
October 18, 2007. http://www.cdc.gov/ncidod/dbmd/
diseaseinfo/groupastreptococcal_t.htm.
2. National Institute of Allergy and Infectious Diseases. Group A
Streptococcal Infections. Accessed October 15, 2007. http://
www.niaid.nih.gov/factsheets/strep.htm.
3. Group A Streptococcal vaccine development: current status
and issues of relevance to less developed countries. Accessed
October 18, 2007. http://www.who.int/child-adolescent-health/
publications/CHILD_HEALTH/DP/Topic_2/paper_3.htm.
4. Schleiss MR. Streptococcal Infection, Group A. Accessed
October 15, 2007. http://www.emedicine.com/ped/topic2702.
htm.
5. Sharma S. Streptococcus Group A Infections. Accessed
October 15, 2007. http://www.emedicine.com/med/topic2184.
htm.
6. Todar K. Todar’s Online Textbook of Bacteriology. Accessed
October 15, 2007. http://www.textbookofbacteriology.net/
7. Ebell MH, Smith MA, Barry HC, Ives K, Carey M. 2000.
The rational clinical examination. Does the patient have strep
throat? JAMA Dec 13;284(22):2912-8.
MEDICAL DIAGNOSTIC LABORATORIES, L.L.C.
Influenza Virus
The Influenza virus is a segmented,
negative sense, single-stranded RNA virus.
The genome, which is approximately 12 to
14 kb in size, encodes for ten proteins (7).
There are three strains of influenza, A, B
and C, that vary from one another in their
pathogenicity. Influenza A strains induce
the most severe infections and are capable of infecting
both humans and animals, while B strain infections are
more restricted to humans and are not as severe (12, 18).
Influenza C strains are associated with very little illness and
are not deemed a public health threat (18). The segmented
viral genome of influenza allows for its constant alteration,
either by point mutation during viral replication (antigenic
drift) or the reassortment of individual genomic segments
(antigenic shift) (5). The process of antigenic drift has allowed
for adamantine-resistant strains of influenza to surface,
rendering both amantadine and rimantadine prophylactic
strategies useless. Medical Diagnostic Laboratories, L.L.C.
offers free reflexive testing for all influenza A specimens
for the determination of resistant strains. Providing this
information to physicians allows patients to be better informed
and to take the precautionary measures to limit its spread.
Figure 1. Summary of influenza typed strains during the 2006-
2007 season. WHO/NREVSS collaborative study (6).
Pathogenesis
The process of antigenic drift occurs slowly and affects
both A and B viral strains. In contrast, antigenic shift occurs
more rapidly and only affects influenza A strains. The
reassortment of these genomic segments occurs as a result
of coinfections within mammals, allowing for the mixing of
viral genomes (19). Within the influenza A viruses, individual
reassortant strains are subtyped by the genomic segments
that are affected, the hemagglutinin (H) and neuraminidase
(N) structural proteins. Due to this, influenza A strains are
typically referred to by the type of both hemagglutinins, of
which there are 16, and neuraminidase, of which there are 9,
incorporated using the HxNx designation. Alterations within
these genomic segments impact the infectious process
directly because these structural proteins are critical to the
virus’ life cycle: hemagglutinin aids the viral entry of host
cells and neuraminidase is required for viral budding from
infected cells (11). Every season influenza viruses are typed
as a method of surveilling for the emergence of new strains
and to aid in determining which strains will be included in the
vaccine for the following season.
Table 1. The death toll following pandemic influenza viral infections.
Pandemic Deaths
United States Worldwide
1918-19 675,000+ 50,000,000+
1957-58 70,000+ 1-2,000,000
1968-69 34,000+ 700,000+
The emergence of recombinant strains, against which
people have little or no immunity, increases the likelihood
of another influenza pandemic (18). In 1918, the worst
influenza pandemic to strike the world occurred, killing
50 million people (Table 1). While pandemics of the 1918
proportion typically do not occur frequently, occurring
approximately every 50 years, the ability of the influenzas
to undergo genetic reassortment increases the likelihood
of developing another highly lethal strain (18). The last
emergence of a novel influenza A strain occurred during the
1968-69 flu season when H3N2 was introduced, inducing a
minor pandemic (15). Recently, the emergence of the H5N1
strain in 1997, often referred to as avian flu due to the fact
that it emerged from chickens, has the world prepared for the
occurrence of a new pandemic (19).
Clinical Significance
The peak of influenza season begins in late December
and extends through early March (6, 18). Infections are
highly contagious and readily transmitted through the
aerosolization of or direct contact with respiratory secretions.
Upper respiratory tract epithelial cells serve as the initial
site of viral infection, resulting in both the desquamation
of ciliated epithelia and the loss of protective mucocilia
(20). The incubation period for influenza is typically 1 to
4 days and is dependent upon the level of exposure (18).
Infected individuals can transmit the virus prior to the onset
of symptoms and continue to shed virus in their nasal
secretions for approximately five days (18). Patients will
present with typical flu-like symptoms, defined by the sudden
onset of fever, headache, chills, malaise and myalgia, all of
which are the by-products of interferon production and the
activation of the body’s natural defense, the antiviral pathway
(10, 18). Individuals with underlying medical conditions or
compromised immunity, including the elderly, are prone to
more severe infection and are at increased risk of developing
pneumonia (8).
www.mdlab.com • 877 269 0090
Treatment
The adamantine class of drugs, which includes amantadine
and ramantadine, are known to be effective at limiting influenza
infections to a subclinical infection in 70% to 90% of the cases
(4). These drugs are effective against A strains of influenza,
targeting the M2 protein that is unique to these strains.
Through both selective pressure and the virus’ propensity
to mutate, adamantine-resistant viral strains have emerged.
Single point mutations of critical residues within the M2 gene
are capable of conferring drug resistance (4, 9). The sudden
rise in transmission of resistant H3N2 strains observed within
the United States has been attributed to the misuse of over-
the-counter amantadine in China following the SARS outbreak
(9). Within the United States, the rate of resistance rose 12.6%
from the 2004 season, a 1.9% incidence rate, to a 14.5%
incidence rate for the first six months of the 2005 season
(Table 2) (4). Currently, the CDC recommends administration
with either zanamivir or oseltamivir within the first two days of
symptom onset when dealing with susceptible strains.
Table 2. Incidence of adamantine resistant influenza isolates.
Adopted from (9).
Vaccine
The number of hospitalizations as a result of influenza
infection is approximately 200,000 annually. Hospitalizations
are typically higher in years with circulating type A strains
(12). Annually, 3,600 deaths occur as a result of infection in
the United States. The elderly and children under the age
of two constitute an especially susceptible segment of the
population (Figure 2) (2, 17, 18). Children under the age of
23 months are at greater risk of hospitalization; determining
an exact rate due to influenza infection is difficult due to the
substantial overlap with respiratory syncytial virus (RSV) (14,
21). Adequate vaccination coverage can markedly decrease
the infective rate (18). There are currently two types of
immunizations available: inactivated influenza vaccine and
the live, attenuated influenza vaccine (LAIV). Both vaccine
strategies contain strains that are antigenically similar to those
predicted to circulate that season, typically including one
H3N2, one H1N1 and one B virus (18). For children under the
age of 9 years, two administrations of vaccine are suggested
for proper effectiveness (1, 16). Several studies have
demonstrated reductions in both direct and indirect medical
costs as a result of more thorough vaccination, particularly in
children and adults 65 years and older (3, 13).
60,000
>85 65-74
50,000 75-84 <65
40,000
30,000
20,000
10,000
0 Infection of human airway epithelium by human and avian strains
1968-69 1968-70 1980-81 1990-91
Pandemic to to to
1979-80 1989-90 2000-2001
Figure 2. Influenza-related deaths by age group.
References:
1. Allison MA, Daley MF, Crane LA, Barrow J, Beaty BL, Allred
N, Berman S, Kempe A. 2006. Influenza vaccine effectiveness
in healthy 6 to 21 month old children during the 2003-2004
season. J Pediatr 149:755-762.
2. Bhat N, Wright JG, Broder KR, Murray EL, Greenberg ME,
Glover MJ, Likos AM, Posey DL, Klimov A, Lindstrom SE,
Balish A, Medina MJ, Wallis TR, Guarner J, Paddock CD,
Shieh WJ, Zaki SR, Sejvar JJ, Shay DK, Harper SA, Cox
NJ, Fukuda K, Uyeki TM. 2005. Influenza-associated deaths
among children in the United States, 2003-2004. N Engl J Med
353:2559-67.
3. Bridges, CB, Thompson WW, Meltzer MI, Reeve GR,
Talamonti WJ, Cox NJ, Lilac HA, Hall A, Klimov A, Fukuda
K. 2000. Effectiveness and cost-benefit of influenza vaccination
of healthy working adults: A randomized controlled trial. JAMA
284:1655-63.
4. Bright, RA, Shay DK, Shu B, Cox NJ, Klimov AI. 2006.
Adamantine resistance among influenza A viruses isolated early
during the 2005-2006 influenza season in the United States.
JAMA 295:891-4.
5. Centers for Disease Control and Prevention. 2006. High levels
of adamantine resistance among influenza A (H3N2) viruses
and interim guidelines for use of antiviral agents--United States,
2005-06 influenza season. MMWR Morb Mortal Wkly Rep 55:44-
6.
6. Centers for Disease Control and Prevention. 2007. Weekly
Report: Influenza Summary Update Week ending May19, 2007-
Week 20.
7. Cheung, TK, Poon LL. 2007. Biology of influenza A virus. Ann
N Y Acad Sci 1102:1-25.
8. Falsey, AR, Walsh EE. 2006. Viral pneumonia in older adults.
Clin Infect Dis 42:518-24.
9. Hayden, FG. 2006. Antiviral resistance in influenza viruses--
implications for management and pandemic response. N Engl J
Med 354:785-8.
10. Horisberger, MA. 1995. Interferons, Mx genes, and resistance
to influenza virus. Am J Respir Crit Care Med 152:S67-71.
11. Lanzrein, M, Schlegel A, Kempf C. 1994. Entry and uncoating
of enveloped viruses. Biochem J 302 ( Pt 2):313-20.
12. Meissner, H. C. 2005. Reducing the impact of viral respiratory
infections in children. Pediatr Clin North Am 52:695-710, v.
13. Nichol, KL, Wuorenma J, von Sternberg T. 1998. Benefits of
influenza vaccination for low, intermediate, and high-risk senior
citizens. Arch Intern Med 158:1769-76.
14. O’Brien, MA, Uyeki TM, Shay DK, Thompson WW, Kleinman
K, McAdam A, Yu XJ, Platt R, Lieu TA. 2004. Incidence of
outpatient visits and hospitalizations related to influenza in
infants and young children. Pediatr 113:585-93.
15. Reichert, TA. 2005. Preparing for the next influenza pandemic:
lessons from multinational data. Pediatr Infect Dis J 24:S228-
31.
16. Ritzwoller, DP, Bridges CB, Shetterly S, Yamasaki K, Kolczak
M, France EK. 2005. Effectiveness of the 2003-2004 influenza
vaccine among children 6 months to 8 years of age, with 1 vs 2
doses. Pediatr 116:153-9.
17. Simonsen, L, Reichert TA, Viboud C, Blackwelder WC,
Taylor RJ, Miller MA. 2005. Impact of influenza vaccination on
seasonal mortality in the US elderly population. Arch Intern Med
165:265-72.
18. Smith, NM, Bresee JS, Shay DK, Uyeki TM, Cox NJ, Strikas
RA. 2006. Prevention and Control of Influenza: recommendations
of the Advisory Committee on Immunization Practices (ACIP).
MMWR Recomm Rep 55:1-42.
19. Sorrell, EM, Ramirez-Nieto GC, Gomez-Osorio IG, Perez DR.
2007. Genesis of pandemic influenza. Cytogenet Genome Res
117:394-402.
20. Thompson, CI, Barclay WS, Zambon MC, Pickles RJ. 2006.
of influenza A virus. J Virol 80:8060-8.
21. Thompson, WW, Shay DK, Weintraub E, Brammer L,
Bridges CB, Cox NJ, Fukuda K. 2004. Influenza-associated
hospitalizations in the United States. JAMA 292:1333-40.
MEDICAL DIAGNOSTIC LABORATORIES, L.L.C.
Respiratory Syncytial Virus (RSV)
Respiratory syncytial virus, (RSV)
is a viral pathogen which most
severely affects infants and the
elderly. Originally isolated from a
chimpanzee with an upper respiratory
tract infection (URTI), the virus was
named Chimpanzee coryza in 1956
and was not named RSV until its isolation from a child
suffering from pneumonia (1). The virus is comprised
of ten proteins, all of which are encoded for by a non-
segmented RNA genome. The name “respiratory
syncytial virus” was assigned based on the pathologic
observation that infected cells form syncytia in vitro
(13). Two viral subtypes are known, A and B, each
having multiple genotypes.
Epidemiology
RSV infections are quite common and their yearly
winter epidemics are widely associated with lower
respiratory tract infections (LRTI), as well as
bronchiolitis and viral pneumonia (4). RSV is the most
common viral infection associated with bronchiolitis.
While infections are typically mild, a small subset will
experience life-threatening complications (4). Recent
evidence suggests possible coinfections with rhinovirus
or human metapneumovirus occur in children with
bronchiolitis. Approximately 40% of RSV infected
children were found to have concomitant otitis media
infections of either bacterial or viral origin (6). Virtually
all children have had at least one RSV infection by their
second birthday and reinfections occur throughout
an individuals’ lifetime (5). While recurrent infections
in older children and adults are typically limited to
the upper respiratory tract (URT), infections during
childhood are most often associated with the LRT
(6). Annually within the United States, RSV-induced
LRTIs occur in 4 to 5 million children and an estimated
18,000 to 75,000 children require hospitalization and
90 to 1,900 succumb to their infection (4, 7). Infection
occurs during the winter months and both the peak and
incidence rates vary geographically within the United
States (Figure 1). While there is no sexual predilection
observed for the mild cases of RSV, the more severe
cases occur twice as frequently in males than females
(6).
Transmission
Due to the fact that infants excrete copious amounts of
virus in their nasal discharges, often at concentrations
greater than 107/mL, combined with the >5 hour
stability of RSV on solid surfaces, physicians and
hospitals have taken measures to limit its spread
through pediatric wards. Medical professionals were
found to serve as vehicles of RSV dissemination and
so recommendations have been made encouraging
the use and frequent change of proper protective
equipment along with quarantine of RSV-infected
children from the general population (6-8).
Figure 1. Seasonal patterns of RSV infections within each region
of the United States.
Clinical Significance
The age of the infant at the time of exposure is a critical
factor, with children six months of age and younger the
most susceptible and most prone to the development
of severe infection. The hospitalization risk for children
was assessed to be 6 cases per every 1,000 child-
years by the World Health Organization (4), with the
number increasing to 11 within the 6 months-and-
under population. Aside from age, several factors, both
environmental and genetic, influence the severity of
RSV infections. Environmentally, daycare attendance,
overcrowded living conditions and contact with
passive smoke all increase the severity of infection.
Genetic predispositions include congenital cardiac
defects, chronic pulmonary disease and relation to an
asthmatic. Premature birth, most notably those under
thirty-five weeks gestation, and immunodeficiency also
serve to predispose infants to more severe infections.
Children with such predispositions typically spend
twice as much time in the hospital, averaging 7 to 8
days per stay as opposed to the typical 3 to 4 days
(6). Severe infections can result in life-threatening
extra-pulmonary complications (Table 1) (9). Higher
morbidity is observed among infants with chronic lung
disease, congenital heart disease and prematurity,
with a reported mortality rate of 3 to 5% (6). While
www.mdlab.com • 877 269 0090
most reports concerning RSV are focused on children,
RSV has more recently been recognized as a cause of
morbidity in elderly populations previously attributed to
influenza (4).
Organs Affected Complications References
Brain Apneas; status (10, 11)
epilepticus
Ventricular
Heart (12-14)
tachycardia;
ventricular fibrilation;
cardiogenic shock;
complete heart block
pericardial
tamponade
Brain, liver Reye’s Syndrome (15)
and kidney
Table 1. Life-threatening extrapulmonary complications that can
result from severe RSV infection (11).
Diagnosis
Procurement of a respiratory specimen is less painful
using a swabbing technique as opposed to a nasal
aspirate. A study published in 2005 compared both
nasal sampling methods. That study concluded that
traditional nasal swabbing techniques were less reliable
than nasal aspirates. A similar study undertaken by
Medical Diagnostic Laboratories, L.L.C. using flocked
swab technology demonstrates the utility of nasal
swabbing for the detection of respiratory pathogens in
both pediatric and adult populations. The flocked nature
of the MDL NasoSwab™ is comprised of Dacron®
fibers arranged in a conical shape. The unique shape
allows for sampling deeper within the nasal passages
unlike traditionally shaped swabs and its flocked nature
captures pathogens more completely. The results of this
study demonstrate greater accuracy associated with
the NasoSwab™, detecting RSV in 15 of 17 samples,
while the traditional nasal wash only identified 9 (Table
2). To serve both pediatric and adult populations,
NasoSwab™ is packaged with individual swabs for use
in either group.
References:
1. Blount RE, Morris JA, Savage RE. 1956. Recovery of
cytopathogenic agent from chimpanzees with coryza. Proceedings
of the Society for Experimental Biology and Medicine Society for
Experimental Biology and Medicine (New York, NY). 92:544-9.
2. Hall CB, Walsh EE, Schnabel KC, Long CE, McConnochie
KM, Hildreth SW, Anderson LJ. 1990. Occurrence of groups
A and B of respiratory syncytial virus over 15 years: associated
epidemiologic and clinical characteristics in hospitalized and
ambulatory children. J Infect Dis 162:1283-90.
3. Martinello RA, Chen MD, Weibel C, Kahn JS. 2002. Correlation
between respiratory syncytial virus genotype and severity of
illness. J Infect Dis 186:839-42.
4. Meissner HC, Long SS. 2003. Revised indications for the use
of palivizumab and respiratory syncytial virus immune globulin
intravenous for the prevention of respiratory syncytial virus
infections. Pediatr 112:1447-52.
5. Glezen WP, Taber LH, Frank AL, Kasel JA. 1986. Risk of primary
infection and reinfection with respiratory syncytial virus. Am J Dis
Children (1960) 140:543-6.
6. Hall CB, Douglas RG, Jr. 1981. Nosocomial respiratory syncytial
viral infections. Should gowns and masks be used? Am J Dis
Children (1960) 135:512-5.
7. Murphy D, Todd JK, Chao RK, Orr I, McIntosh K. 1981. The
use of gowns and masks to control respiratory illness in pediatric
hospital personnel. J Pediatr 99:746-50.
8. Gala CL, Hall CB, Schnabel KC, Pincus PH, Blossom P,
Hildreth SW, Betts RF, Douglas RG Jr. 1986.The use of eye-
nose goggles to control nosocomial respiratory syncytial virus
infection. JAMA 256:2706-8.
9. Eisenhut M. 2006. Extrapulmonary manifestations of severe
respiratory syncytial virus infection--a systematic review. Crit
Care (London, England) 10:R107.
10. Kho N, Kerrigan JF, Tong T, Browne R, Knilans J. 2004.
Respiratory syncytial virus infection and neurologic abnormalities:
retrospective cohort study. J Child Neurol 19:859-64.
11. Sweetman LL, Ng YT, Butler IJ, Bodensteiner JB. 2005.
Neurologic complications associated with respiratory syncytial
virus. Pediatr Neurol 32:307-10.
12. Armstrong DS, Menahem S. 1993. Cardiac arrhythmias as
a manifestation of acquired heart disease in association with
paediatric respiratory syncitial virus infection. J Pediatr Child
Health 29:309-11.
13. Thomas JA, Raroque S, Scott WA, Toro-Figueroa LO, Levin
DL. 1997. Successful treatment of severe dysrhythmias in
infants with respiratory syncytial virus infections: two cases and a
literature review. Crit Care Med 25:880-6.
14. Hutchison JS, Joubert GI, Whitehouse SR, Kissoon N. 1994.
Pericardial effusion and cardiac tamponade after respiratory
syncytial viral infection. Pediatr Emerg Care 10:219-21.
15. Griffin N, Keeling JW, Tomlinson AH. 1979. Reye’s syndrome
associated with respiratory syncytial virus infection. Arch Dis
Childhood 54:74-6.
16. MacFarlane P, Denham J, Assous J, Hughes C. 2005. RSV
testing in bronchiolitis: which nasal sampling method is best?
Arch Dis Childhood 90:634-5.

Saline Nasal Wash NasoSwab™

 
Positive Negative Total Positive Negative Total
Abnormal 9 8 17 15 2 17
Positive
by PCR

Normal 0 11 11 0 12 12
Total 9 19 28 15 14 29
Table 2. Comparison of two collection methods for the detection of RSV in nasal specimens in a blinded study. The standard
saline nasal wash was compared to the NasoSwab™ method of collection. The NasoSwab™ collection method proved to be
more sensitive, having an 88.2% detection rate as opposed to the 52.9% obtained with the nasal wash method.
MEDICAL DIAGNOSTIC LABORATORIES, L.L.C.
Human Parainfluenza Viruses
The Human Parainfluenza Viruses
are closely related respiratory tract
pathogens capable of inducing a
varying spectrum of disease from
the common cold to influenza-like
illness or pneumonia. Although
croup is often the most common
and severe manifestation, 30% to
50% of upper respiratory tract infections (URTI) are
further complicated by otitis media (3). There are four
serotypes, designated HPIV-1 through HPIV–4 and
two subtypes, HPIV-4a and HPIV-4b. These viruses
are members of the Paramyxoviridae family, with
HPIVs 1 and 3 members of the genus and HPIVs 2,
4a and 4b members of rubulavirus genus, which also
contains the mumps virus (1, 2, 5). A common cause
of respiratory tract infections in children, surpassed
only by respiratory syncytial virus (RSV), the HPIVs
are capable of inducing infections throughout an
individuals’ lifetime. Although there is a high rate of
recurrent infection, overt symptomology appears to be
limited to the primary infection, which typically occurs
by the age of five years, while subsequent infections
are either asymptomatic or less severe (5).
Pathology
The parainfluenza viruses are negative-sense, single-
stranded RNA viruses whose infectious outcome is
dependent upon the strain. Types 1 and 2 follow an
autumn/winter seasonal distribution and have been
associated with children between the ages of two and
six (5). Type 3 demonstrates no seasonal distribution,
with detection both sporadically and year-round. Within
the United States, HPIV-3 is considered endemic and
affects mostly children under the age of one. Little is
known about the seasonal distribution of HPIV-4, in part
because it is believed to be limited to mild URTI (1).
More serious infections, including aseptic meningitis,
parotitis and otitis media, can occur though only rarely.
The immunocompromised are at greater risk of more
severe and persistent infection and have demonstrated
increased viral shedding. HPIV-3 infections follow a
spring/summer seasonal trend and outbreaks tend to
last longer than do those associated with either HPIV-
1 or HPIV-2 (Figure 1) (2). The incubation period
lasts an average of 1 to 10 days (5). The lack of overt
symptoms within adult populations increases the
likelihood of transmission within the more susceptible
pediatric and juvenile populations and occurs primarily
through the inhalation of infected respiratory droplets,
direct contact of mucous membranes of the eyes,
mouth or nose with infected respiratory secretions or
through contact with infected inanimate surfaces (1,
5). As a result, outbreaks are increased in situations
where close person-to-person contact occurs more
frequently, as with hospital nurseries, pediatric wards
and schools (2).
Figure 1. Increased prevalency of HPIV-3 infections during the
spring and autumn seasons. Adapted from (1).
Symptoms
While the seasonality among the various HPIV strains
might aid in their diagnosis, often the initial clinical
presentation makes diagnosis from other respiratory
pathogens difficult. While croup diagnosis during later
stages of infection are quite distinguishable from other
respiratory infections, characterized by a harsh, seal-
like barking cough, the initial presentation is analogous
to that of the common cold (2). Croup resulting from
(Table 1) HPIV infection is the most prevalent cause
of annual hospitalization for the 2 to 6 years age
group of children (Table 1). The annual hospitalization
rate within the United States for HPIV-1 is 5,800 to
28,900 and 1,800 to 15,600 for HPIV-2. HPIV-3, a less
common cause of croup than HPIV-1 or –2, is more
likely to cause pneumonia or bronchiolitis in pediatric
populations under the age of six months and is a
common cause of hospitalization within this age group.
Such rates are similar to that observed with RSV,
which has an average annual hospitalization number
of 8,700 to 52,000 (2, 3, 5) (Table 2). HPIV-4, the least
characterized of the parainfluenza viruses, has thus far
only been associated with mild upper respiratory tract
infections (URTI) (Table 2).
www.mdlab.com • 877 269 0090
Table 1. Infectious outcomes of HPIV subtype infection. Adapted
from (4).
Infectious Outcome Viral Type
Minor URTI 1, 3, 4
Bronchitis 1, 3
Pneumonia 1, 3
Croup 1, 2, 3
Vaccine
Presently, there are no vaccines available to confer
protection against infection with any of the HPIV strains;
children are totally reliant upon the passive immunity
transferred to them from their mothers. Presently, there
are two HPIV-3 and one HPIV-1 vaccine strategies
currently under development. These strategies have
thus far been hampered by an inability to balance the
viral replication required within the child upon vaccination
for the induction of a proper immune response in the
presence of maternally conferred antibodies (3).
Diagnosis
HPIV infections during childhood are quite common, as
revealed by retrospective serological surveys that have
shown 90% or greater of children aged five years and
under have been exposed to HPIV-3 and approximately
75% of this population has been exposed to HPIV-1
and HPIV-2 based on the presence of strain-specific
antibodies (3). The fact that these serotypes display
a marked age differential, with respect to infection,
makes the ability to differentiate among these species
necessary from a diagnostic standpoint. Two methods,
direct detection of the virus and indirect methods,
are considered acceptable. Direct detection methods
encapsulate those methods that directly identify viral
elements, whether DNA, RNA or protein, while indirect
methods typically infer an infection has occurred based
upon the presence of viral-specific antibodies in the
patient’s serum.
Indirect Methods:
IFA/ ELISA
Both the IFA and ELISA methods of indirect detection
base the occurrence of infection upon the presence of
viral-specific antibodies within patient sera. Such assays
can be either for IgM (early stage infection) or IgG (late
stage infection) and are subject to the sensitivity and
specificity of the antibodies employed; deficiencies in
either parameter can increase the likelihood of false
positive or false negative determinations. The utilization
of such methods, at least with respect to identifying the
cause of respiratory infections, is whether or not the
patient, particularly pediatric patients, have immune
systems capable of producing either antibody class.
(Figure 1) demonstrates this point with respect to HPIV-
3 detection whereby viral antigens were undetectable
within the 1 year and younger age group despite the
clear ability to isolate the virus from these individuals (1).
Diagnoses of HPIV-4 infections are further complicated
due to their antigenic cross-reactivity with the mumps
(2).
Direct Methods:
Immunofluorescent Staining
Respiratory secretions can be smeared along a glass
slide for incubation with viral-specific antibodies
and visualized directly through immunofluorescent
microscopy. The limitations associated with such assays
are the need for specialized, expensive equipment,
the unavailability or poor specificity of commercially
available antibodies and the high degree of time and
skill required to process such samples.
Polymerase Chain Reaction (PCR)
PCR reactions, when designed and validated properly,
can be a fast and efficient means of diagnosing infections.
This method involves the repeated amplification of
viral-specific genetic elements that can often serve to
speciate among the conserved HPIV viruses.
MDL has developed a new method of respiratory tract
sampling, the NasoSwab™, and a series of Real-Time
PCR assays to aid physicians in discerning the causative
agent responsible for these overlapping symptoms.
The novel “flocked-swab” technology employed by
the NasoSwab™ represents a less invasive manner
of sampling the upper respiratory tract whose particle
retention, as measured by both level, of specificity and
sensitivity by an independent, blinded study, matches
or exceeds the levels obtained with nasal washes.
The use of specialized stabilization media allows for
room temperature storage of specimen that serves to
preserve the integrity of the pathogen better than does
the immediate freezing of nasal wash specimen.
Table 2. Incidence of parainfluenza co-infection with other respiratory
pathogens. Adapted from (6).
Virus combinations No. of patients
(% with DRVI)
Influenzavirus A, picornavirus 10 (14.9)
Picornavirus, coronavirus 10 (14.9)
Adenovirus, picornavirus
Influenzavirus A, RSV
7 (10.4)
7 (10.4)
RSV, parainfluenzavirus 6 (9.0)
RSV, picornavirus 4 (6.0)
RSV, coronavirus 4 (6.0)
Parainfluenzavirus, coronavirus 4 (6.0)
Influenzavirus A, adenovirus 4 (6.0)
Influenzavirus A, influenzavirus B 3 (4.5)
Influenzavirus A, parainfluenzavirus 2 (3.0)
Influenzavirus A, coronavirus 2 (3.0)
Influenzavirus B, RSV 2 (3.0
Influenzavirus A (H1), influenzavirus A (H3) 1 (1.5)
Influenzavirus B, adenovirus 1 (1.5)
Influenzavirus B, picornavirus 1 (1.5)
Influenzavirus B, coronavirus 1 (1.5)
Parainfluenzavirus, adenovirus 1 (1.5)
Parainfluenzavirus, picornavirus 1 (1.5)
Influenzavirus A, RSV, coronavirus
Total
1 (1.5)
67 (100)
References:
1. Centers for Disease Control and Prevention: Respiratory and Enteric Virus Branch.
Last updated on June 6, 2006. Human Parainfluenza Viruses (Common cold and
croup). Accessed October 15, 2007. http://cdc.gov/ncidod/dvrd/revb/respiratory/
hpivfeat.htm.
2. Parainfluenza Virus Infections. Accessed October 15, 2007. http://www.merck.com/
mmpe/sec14/ch188/ch188e.html
3. The World Health Organization: Initiative for Vaccine Research. Parainfluenza
viruses. Accessed October 15, 2007. http://www.who.int/vaccine_research/
diseases/ari/en/index2.html.
4. Parainfluenza Viruses. Accessed October 15, 2007. http://virology-online.com/
viruses/Parainfluenza.htm.
5. N. Narayan, M. D. Parainfluenza Virus. Accessed October 15, 2007. http://
pathmicro.med.sc.edu/virol/para-rsv-aden.htm
6. Drews, A. L., R. L. Atmar, W. P. Glezen, B. D. Baxter, P. A. Piedra, and S. B.
Greenberg. 1997. Dual respiratory virus infections. Clin Infect Dis 25:1421-9.
MEDICAL DIAGNOSTIC LABORATORIES, L.L.C.
Human Metapneumovirus
Human Metapneumovirus (hMPV) is a relatively
recently identified cause of respiratory tract infection.
Identified in 2001 by a Dutch group performing a
retrospective evaluation of nasal secretions collected
over a twenty year period, hMPV was found to be
present in twenty of the twenty-eight samples that
were subjected to random-priming Polymerase Chain
Reaction (PCR) amplification (24). Sequence analysis
confirmed the isolation of a heretofore unknown
respiratory pathogen and also demonstrated it to be
most closely related to avian pneumovirus, a member
of the genus Metapneumovirus (24). Despite its
recent identification, a screen of banked human sera
revealed that hMPV did not recently “jump” into human
populations but was circulating
among them for at least fifty years
(24). Sera analyses also revealed
that a high rate of infection is
associated with hMPV, with nearly
all children age five years having
had prior exposure to the virus
(13) (Table 1).
hMPV RSV
% Positive % Positive
Age (# positive / # (# positive / #
tested) tested)
< 4 months 67 (6/9) 78 (7/9)
4 Months to 1 Year 11 (3/27) 48 (13/27)
1-2 Years 44 (17/39) 54 (21/39)
2-5 Years 76 (19/25) 87 (21/25)
Table 1. Seroprevalence rates for hMPV and RSV in juvenile
populations. Adapted from (7).
Pathology
The genome of hMPV is comprised of negative-sense,
single-stranded RNA whose genes are arranged in
a manner similar to that of its most closely related
human pathogen, respiratory syncytial virus (RSV),
another pneumovirus. This relatively high degree of
homology between hMPV and RSV, combined with
their overlapping epidemiologies and symptomologies
and disease courses, complicates diagnoses. However,
reverse transcriptase PCR-based (RT-PCR) diagnostic
methodologies capable of exploiting the sequence
variations between these related viruses do exist and
are considered the preferred method of differentiation
(5). While the bulk of hMPV pathological information
has been obtained from animal studies, two separate
reports from analyses of human lung biopsies have
shown the virus is capable of inducing both chronic
and acute inflammatory changes of the airways and
organizing lung injuries (2, 23, 25). This common
cause of pediatric bronchiolitis is believed to serve as
a risk factor for the onset of childhood asthma and has
been shown to exacerbate congestive heart disease
and chronic obstructive pulmonary disease (COPD) in
adult populations (9, 10, 26).
Clinical Significance
Human metapneumovirus infections are responsible
for a large number of lower respiratory tract infections
(LRTI) in pediatric populations, second only to RSV
with respect to the number of bronchiolitis cases (3, 4,
17, 18). Various studies report an average incidence
rate between 5% and 15%, though higher rates have
been documented in individual reports (3, 6, 8, 14,
16). Children age 4 months and younger appear to be
the most susceptible population, with relatively high
frequencies reported for all children 2 years of age
and under (11) (Figure 1). The virus is transmitted
via inhalation of infected respiratory droplets or
through direct contact with respiratory secretions or
contaminated surfaces (15). Symptoms of infection are
indistinguishable from those following RSV infection
and range from mild upper RTI to more severe LRTI that
include bronchiolitis and pneumonia (3). British studies
demonstrated that a more severe bronchiolitis was
associated with hMPV/RSV co-infections suggesting
hMPV may serve as a determinant of RSV disease
severity (12, 21). A winter/spring seasonality has
been reported within temperate zones, although some
summer outbreaks have been reported worldwide (20)
(Figure 2).
Figure 1. Age distribution of hMPV infections as determined by a
Real-Time PCR-based diagnostic method in juvenile populations.
Adapted from (11).
www.mdlab.com • 877 269 0090
Diagnosis
The fastidious nature of the virus coupled with the
extremely slow nature of direct culturing methods
prevents this method to be employed as a diagnostic
tool. Currently, RT-PCR methods are considered
to be the fastest and most reliable means of clinical
evaluation, offering the added benefit of being able to
differentiate between a host of respiratory pathogens
that present initially with overlapping symptomologies.
Serological testing thus far has only proved useful
in retrospective studies as the high rate of infections
within pediatric populations precludes their use as a
diagnostic tool.
18%
16%
14%
12%
10%
8%
6%
4%
2%
0%
May 05
May 06
Aug 05
Sep 05
Nov 05
Dec 05
Aug 06
Sep 06
Feb 05
Mar 05
Feb 06
Mar 06
Jan 05
Jun 05
Jan 06
Jun 06
Apr 05
Oct 05
Apr 06
Oct 06
Jul 05
Jul 06
Figure 2. Seasonal distribution of hMPV infections. Black bars
show the number of tested positive specimens while the black line
shows the percent positivity for hMPV. Adapted from (19).
Treatment
Antiviral therapies for viral respiratory infections, with
the exception of influenza virus, have not shown much
efficacy (13, 17). The ineffectiveness of such therapy
is believed to be due to the induction of high levels
of inflammation within the airway epithelia following
either hMPV or RSV infection. Ribaviran therapy has
been employed for severe cases of hMPV and RSV
infections with some reported success but remains a
controversial form of treatment (1). Presently, the most
promising hMPV therapy currently under development
is the prophylactic treatment of high-risk children with
neutralizing antibodies directed against hMPV’s F
protein (22). Presently, there are no available vaccines
for the prevention or limitation of hMPV infection.
1. Committee on Infectious Diseases. 1996. Reassessment of
the indications for ribavirin therapy in respiratory syncytial virus
infections. American Academy of Pediatrics Committee on
Infectious Diseases. Pediatrics 97(1):137-40.
2. Bao, X, Liu T, Spetch L, Kolli D, Garofalo RP, Casola A. 2007.
Airway epithelial cell response to human metapneumovirus
infection. Virol.(2007). doi:10.1016/j.virol.2007.06.023
3. Boivin G, De Serres G, Cote S, Gilca R, Abed Y, Rochette L,
Bergeron MG, Dery P. 2003. Human metapneumovirus infections
in hospitalized children. Emerg Infect Dis 9:634-40.
4. Chano F, Rousseau C, Laferriere C, Couillard M, Charest
H. 2005. Epidemiological survey of human metapneumovirus
infection in a large pediatric tertiary care center. J Clin Microbiol
43:5520-5.
5. Deffrasnes C, Hamelin, Boivin G. 2007. Human metapneumovirus.
Semin Respir Crit Care Med 28:213-21.
6. Dollner H, Risnes K, Radtke A, Nordbo SA. 2004. Outbreak of
human metapneumovirus infection in norwegian children. Pediatr
Infect Dis J 23:436-40.
7. Ebihara T, Endo R, Kikuta H, Ishiguro N, Ishiko H, Kobayashi
K. 2004. Comparison of the seroprevalence of human
metapneumovirus and human respiratory syncytial virus. J Med
Virol 72:304-6.
8. Esper F, Martinello RA, Boucher D, Weibel C, Ferguson D,
Landry ML, Kahn JS. 2004. A 1-year experience with human
metapneumovirus in children aged <5 years. J Infect Dis
189:1388-96.
9. Falsey AR, Erdman D, Anderson LJ, Walsh EE. 2003. Human
metapneumovirus infections in young and elderly adults. J Infect
Dis 187:785-90.
10. Garcia-Garcia ML, Calvo C, Casas I, Bracamonte T, Rellan
A, Gozalo F, Tenorio T, Perez-Brena P. 2007. Human
metapneumovirus bronchiolitis in infancy is an important risk
factor for asthma at age 5. Pediatr Pulmonol 42:458-64.
11. Gray GC, Capuano AW, Setterquist SF, Erdman DD, Nobbs
ND, Abed Y, Doern GV, Starks SE, Boivin G. 2006. Multi-
year study of human metapneumovirus infection at a large US
Midwestern Medical Referral Center. J Clin Virol 37:269-76.
12. Greensill J, McNamara PS, Dove W, Flanagan B, Smyth RL,
Hart CA. 2003. Human metapneumovirus in severe respiratory
syncytial virus bronchiolitis. Emerg Infect Dis 9:372-5.
13. Kahn JS. 2006. Epidemiology of human metapneumovirus. Clin
Microbiol Rev 19:546-57.
14. Maggi F, Pifferi M, Vatteroni M, Fornai C, Tempestini E,
Anzilotti S, Lanini L, Andreoli E, Ragazzo V, Pistello M,
Specter S, Bendinelli M. 2003. Human metapneumovirus
associated with respiratory tract infections in a 3-year study of
nasal swabs from infants in Italy. J Clin Microbiol 41:2987-91.
15. Mahalingam S, Schwarze J, Zaid A, Nissen M, Sloots T, Tauro
S, Storer J, Alvarez R, Tripp RA. 2006. Perspective on the host
response to human metapneumovirus infection: what can we
learn from respiratory syncytial virus infections? Microbes Infect
8:285-93.
16. McAdam AJ, Hasenbein ME, Feldman HA, Cole SE, Offermann
JT, Riley AM, Lieu TA. 2004. Human metapneumovirus in
children tested at a tertiary-care hospital. J Infect Dis 190:20-6.
17. McIntosh K, McAdam AJ. 2004. Human metapneumovirus--an
important new respiratory virus. N Engl J Med 350:431-3.
18. Mullins JA, Erdman DD, Weinberg GA, Edwards K, Hall
CB, Walker FJ, Iwane M, Anderson LJ. 2004. Human
metapneumovirus infection among children hospitalized with
acute respiratory illness. Emerg Infect Dis 10:700-5.
19. Pabbaraju K, Wong S, McMillan T, Lee BE, Fox JD.
2007. Diagnosis and epidemiological studies of human
metapneumovirus using real-time PCR. J Clin Virol 40(3):186-
92.
20. Peiris JS, Tang WH, Chan KH, Khong PL, Guan Y, Lau YL,
Chiu SS. 2003. Children with respiratory disease associated
with metapneumovirus in Hong Kong. Emerg Infect Dis 9:628-
33.
21. Semple MG, Cowell A, Dove W, Greensill J, McNamara PS,
Halfhide C, Shears P, Smyth RL, Hart CA. 2005. Dual infection
of infants by human metapneumovirus and human respiratory
syncytial virus is strongly associated with severe bronchiolitis. J
Infect Dis 191:382-6.
22. Skiadopoulos MH, Biacchesi S, Buchholz UJ, Amaro-
Carambot E, Surman SR, Collins PL, Murphy BR. 2006.
Individual contributions of the human metapneumovirus F, G,
and SH surface glycoproteins to the induction of neutralizing
antibodies and protective immunity. Virology 345:492-501.
23. Sumino KC, Agapov E, Pierce RA, Trulock EP, Pfeifer JD,
Ritter JH, Gaudreault-Keener M, Storch GA, Holtzman NJ.
2005. Detection of severe human metapneumovirus infection
by real-time polymerase chain reaction and histopathological
assessment. J Infect Dis 192:1052-60.
24. van den Hoogen BG, de Jong JC, Groen J, Kuiken T,
de Groot R, Fouchier RA, Osterhaus AD. 2001. A newly
discovered human pneumovirus isolated from young children
with respiratory tract disease. Nat Med 7:719-24.
25. Vargas SO, Kozakewich HP, Perez-Atayde AR, McAdam AJ.
2004. Pathology of human metapneumovirus infection: insights
into the pathogenesis of a newly identified respiratory virus.
Pediatr Dev Pathol 7:478-86; discussion 421.
26. Williams JV, Crowe JE, Enriquez R, Minton P, Peebles RS,
Hamilton RG, Higgins S, Griffin M, Hartert TV. 2005. Human
metapneumovirus infection plays an etiologic role in acute
asthma exacerbations requiring hospitalization in adults. J Infect
Dis 192:1149-53.
 Medical Diagnostic Laboratories, L.L.C.
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www.mdlab.com • Toll Free 877 269 0090