Ministry of Health of the Republic of Moldova

PUBLIC INSTITUTION “NICOLAE TESTEMIŢANU”

STATE UNIVERSITY OF MEDICINE AND PHARMACY

OF THE REPUBLIC OF MOLDOVA

FACULTY OF MEDICINE No 2

Department of Microbiology, Virology and Immunology

DIPLOMA THESIS

Microbiology and laboratory diagnosis of whooping cough

Student:
Wala Nassar
6th year, group 1046

Scientific Adviser:
As. Dr. Cojocari Daniela

Chisinau, 2016

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 CONTENTS
I. Introduction......................................................................4
1.1. Actuality of the theme.................................................................................4
1.2. Goals and objectives of the thesis…………………………………………5
1.3. The scientific importance of the results of this study. .................................5
II. Bibliographical analysis of the theme……………………..7
2.1 . Historical aspects of whooping cough ………………………………………7

2.2. Epidemiology of Whooping cough ………………………………………….9

2.2.1. Source of infection. …………………………………………………………9
2.2.2. Transmission………………………………………………………………9
2.2.3. Statistics…………………………………………………………………11
2.2.4. The situation in Moldova…………………………………………………14
2.3. Whooping cough. Classification of the causative agent (family, genus, sp.)..15
2.3.1. Morphology of B. pertussis………………………………………………16
2.3.2. Cultural characteristics…………………………………………………….16
2.4. Virulence factors……………………………………………………………..17

2.4.1. Pathogenesis…………………………………………………………………….....18

2.5. Clinical manifestation ……………………………………………………………21

2.5.1. Complications…………………………………………………………………23
2.6. Immunity……………………………………………………………………. 24
2.7. Laboratory diagnosis…………………………………………………………25
2.7.1. Specimens. Transportation.......................................................................... 25
2.7.2. Direct microscopy. ………………………………………………………..26
2.7.3. Bacteriological method of diagnosis............................................................27

2.7.4. Modern techniques of cultivation of Bordetella...........................................27

2.7.5. Serological method of diagnosis..................................................................28

2.7.6. Differential diagnosis...............................................................................30

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2.8. Prevention…………………………………………………………………..30

2.8.1. Pertussis vaccine………………………………………………………….32

2.9. Treatment of whooping cough........................................................................34

III. MATHERIALS AND METHODS OF RESEARCH……………………....36
IV. RESULTS OF THE STUDY……………………………………………….38
V. CONCLUSIONS……………………………………………………………..39

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 I. INTRODUCTION

1.1 Actuality of the theme

Whooping cough is one of the most popular infectious diseases in the world.
It is an acute and highly contagious infection, which mainly affects the upper
respiratory system. Whooping cough is predominantly a pediatric disease. The
mortality and the incidence is highest in the first year of life.
The actuality of this infection is based on the fact that it is caused by severe
clinical symptoms and also affects mainly children. Left untreated complications
can be fatal.
Pertussis is also known as whooping cough because of the particular noise
during the coughing crisis. Whooping cough is caused by Bordetella, an aerobic
coccobacillus, which produce a toxin that paralyzes respiratory cells and causes
inflammation. The disease begins like a common cold but then develops into a
severe cough which can last for weeks. It is a very contagious disease which causes
coughing and it can be so severe that leads to vomiting and aspiration of vomit.
The name of the disease, whooping cough, comes from the sound made by the
sudden inhalation after a sustained cough. The disease can be severe at any age,
especially in children, to whom it can cause apnea, or brief pauses in breathing.

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 Also the current developments in different countries suggest a possible
correlation to some case autism. One of the hypotheses is that it can be a
consequence of vaccination against pertussis. First of all, there have not yet been
enough studies to confirm or infirm this hypothesis. One of the aspects relates to
the vaccine being a combination for two other infections – diphtheria and tetanus.
Nonetheless this is a very sensitive issue, this is why it is worth the discussions.

1.2 Goals and objectives of the thesis

Goal:
The goal of this thesis is to assess the current situation of the diagnosis of
whooping cough. This includes all the existing classic and modern diagnosis
methods, predominant practices both in Moldova and in other countries.
Objectives:
1. To establish the causal agent according to the existing theories on

whooping cough;
2. To determine the morphological and biological properties of pertussis;

3. To list and describe the classic and modern diagnosis methods of pertussis;

4. To describe the prophylaxis methods currently used and assess their

efficiency;
5. To describe possible complications if not diagnosed and treated timely;
6. To conduct a compared statistical analysis of incidence of whooping

cough;
7. To study the possible connection of the pertussis vaccine with cases of
autism in children.

1.3 The scientific importance of the results of this study

This research provides general overview of pathogenesis, clinical symptoms,
and the array of diagnostic methods use for whooping cough. It also shows that in
spite of all measures taken to combat this infection, it still present in our days. The
review of diagnostic methods and the analysis of their efficiency allows for better
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understanding of possible measures that would allow to better prevent, diagnose
and treat this disease.
It will also allow us to understand the suspected correlation between the
whooping cough vaccine and the incidence autism in children.

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 II. Bibliographical analysis of the theme
Historical aspects of whooping cough

Infection is characterized by a harrowing cough feature that lasts weeks and

months. Many centuries parents have struggled to protect children against of
pertussis. How much the disease has affected mankind is not known.
In the age of 1414 was for the first time mentioned about epidemics of
whooping cough in France. Clinical symptoms of pertussis were described in
1540. The first description of the infection was made by French doctor Guillaume
Baillou during a n outbreak in Paris in 1578.

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 Paris and Rome were terrible epidemic of pertussis in 1695. In Scandinavia, it
was an epidemic of fifteen years in the half of the eighteenth century. In England
between 1858 and 1865 died more than 120,000 people.
Pertussis has been something ordinary so many centuries. Each European
country has created its own popular name for the disease. In 1679 year Sidenhem
named the disease Whooping cough or pertussis means intensive cough. In Italy
the infection was named Tosse canina (dog’s barking). In England pertussis was
named chincough and it means suffocating cough.
In 1900 B. pertussis, causal agent was first found microscopically in mucus of
a patient with whooping cough. Gengou and Jules Bordet identified the small
ovoid bacillus and succeeded in cultivating it in a complex medium.
Doctors did not know how to handle or deal with disease. Using leeches to
treat patients and incision was widely practiced therapy for pertussis in the
seventeenth century and a doctor of that era. Purgatives were administered to
victims with pertussis. Many plants were used as medicine for whooping cough.
The first experimental vaccine was created in 1908 year from killed culture of
Bordetella pertussis. Experimental vaccination of people against of whooping
cough is begun in 1926. World-wide vaccination with whole cell vaccine is started
in 1945-1960. Because of this infection cases started to decline
Even though doctors have not been able to prevent or cure pertussis,
morbidity and mortality have declined continuously since the mid-nineteenth
century. One explanation for the decline in the mortality rate of this disease may be
that the population of Europe and the United States achieved a certain degree of
natural resistance. Another reason for the decline of whooping cough before using
the vaccine was immense improvement of living standards. Sanitation, nutrition
and better housing and health care have led to a better general health of the infant
population.
The introduction of antibiotics during the Second World War, has had
dramatic effect on pertussis. The successful use of antibiotics has given children a
much better chance to survive pertussis
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 2.2. Epidemiology of Whooping cough
Pertussis is a highly communicable disease. Pertussis occurs in all months.
Whooping cough is a typical anthroponouse disease, the nature of reservoir of
which is human. Infection is predominantly a pediatric disease. The incidence and
mortality is highest in the first year of life, because maternal antibodies do not give
protection against the disease. Immunization has to be started early. The disease is
worldwide in distribution and it occurs in epidemic form periodically but the
infection is never absent from any community.
2.2.1. Source of infection.
The main source of infection in whooping cough is sick person, patients in the
early stage of disease. Infected people are most contagious up to about 2 weeks
after the cough begins. Infants are more likely to become infected with pertussis. It
may be frequent from their siblings than from their mothers. The main source
consists of adults with atypical or undiagnosed infection, who can transmit the
infection to infants and children.
2.2.2. Transmission
Microorganisms that cause pertussis are carried in the nose, lungs, throat. To
catch it is necessary to inhale the bacteria that somebody else has coughed out. It is
necessary to inhale thousands of bacteria to be infected. The same is important not
to have immunity to whooping cough to get it easily. There are other things that
make people more
susceptible to whooping cough. People who suffer from asthma, viral cold,
pneumonia, bronchitis also seem more susceptible to whooping cough. This make
the diagnosis of whooping cough more difficult. [17]
People with pertussis usually spread the disease to another person by
coughing or sneezing or when spending a lot of time near one another where you
share breathing space. Many babies who get pertussis are infected by older
siblings, parents, or caregivers who might not even know they have the disease.
Infected people are most contagious up to about 2 weeks after the cough begins.
Antibiotics may shorten the amount of time someone is contagious.
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 If generalize, we can say that the infection is spread all over the world. The
route of transmission is airborne and droplets. Pertussis is not of a seasonal nature,
but peak of frequency is August-September. The epidemiological cycle is 3-4
years. Infectiousness is 100% in non-vaccinated persons.
Whooping cough is commoner in the female in the male at all ages. The
secondary attack rates are highest in close house hold contacts. For adults and
adolescents disease often is atypical. They can be source of infection in children.
Chronic carriers are not known. Natural infection confers protection, but it may not
be permanent and second attack have been reported.
This is a milder disease and the incidence varies in different countries.

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 1.2.3. Statistics

Figure 1. The distribution of whooping cough cases worldwide, 2015.

Source: The Global Health Program at the Council on Foreign Relations, USA.

From the figure above we notice that for 2015 the outbreaks of whooping
cough are located in the more developed countries and regions: South-West
Europe, North America (with a higher incidence in the Washington-New-York
region on the East coast) and also the southern Australia. This may be explained by
a higher health education and awareness in these regions, leading to a better
identification and reporting of cases due to differentiated diagnosis methods that
allow to discriminate this infection from other respiratory infections.

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Table 1. Incidence of pertussis (number of reported cases)

Country/Region 2014 2013 2012 2011 2010 2000 1980

198235500
GLOBAL 172904000 161889000 249746000 171740000 160710000 190475000 0
EUROPE 43774000 28170000 57539000 34436000 28212000 53675000 90546000
Bulgaria 52 89 102 46 54 106 154
Germany 12260 258
Israel 1509 1394 2730 2345 1244 452 19
Italy 225 302 187 2543 16643
Moldova 188 115 92 102 31 169
Romania 87 57 82 86 29 493 11441
Russia 4705 4510 7220 4733 4795 29983
Turkey 68 33 18 242 48 510 1520
Ukraine 2286 2937 1067 2243
USA 32784 28532 47693 18610 27410 7867 1730

Source: WHO vaccine-preventable disease monitoring system, 2015 global summary

The trend that can be observed from the table above reflects a decreasing
trend of pertussis incidence globally starting with 2000. But at the same time an
increase is noted for European countries as well as other regions. This increase is
significant for Europe where the incidence increases with more than one third (as
explained for fig.1)

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Figure. Incidence of pertussis based on the number of reported cases

Source: WHO vaccine-preventable disease monitoring system, 2015 global summary

This chart show several countries from all over the world, but also from the
region according to the proximity to Moldova. Higher numbers of cases of
pertussis infection for some of the countries (such as the USA) are based on higher
total number of population.

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 2.2.4. The situation in Moldova
At the same time, in 2014, an increase of the pertussis morbidity to 4,61%000
was observed (objective - under 1,5%000), 70,2% of infected persons having not
been vaccinated for whooping cough.

Table: Number of serological investigations (2012-2014)

Method of 2012 2013 2014

Samples Investigations Samples Investigations Samples Investigations
diagnosis
Pertussis 103 155 154 290 258 507

Source: National Center for Public Health of Moldova: Annual Report on Population
Health for 2014

This year, for pertussis 258 persons have been investigated, 507 investigations have been
carried out to determine the level of IgM immunoglobulins IgM, 135 of them being positive and
252 for IgG – 139 positive. The number of investigations in comparison with 201 and 2013 have
a significantly increasing tendency. The level of both IgM immunoglobulins and IgG during
these years is reported to the number of investigations carried out.

Fugure: Number of investigations and cases of pertussis (2012-2014)

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 2.3. Whooping cough.
Classification of the causative agent (family, genus, sp.)

The genus Bordetella consists of very small coccobacilli and are parasite of
human. It belongs to:

Family: Halobacteriaceae

Genus: Bordetella

Species: Bordetella pertussis (whooping cough agent)

Bordetella parapertussis (parapertussis agent)

Bordetella bronhiseptica (pneumonia, bacteremia)

Bordetella avium (which causes respiratory disease in turkeys)

Bordetella holmensii (isolated from blood cultures)

Bordetella hinzii (isolated from respiratory sampled)

Bordetella trematum (skin and ear infections)

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 2.3.1Morphology of B. pertussis

B. pertussis is a very small Gram-negative aerobic coccobacillus that appears

singly or in pairs. It is non-sporing and non-motile. It is capsulated. The capsule
can be demonstrated by special stains. Freshly isolated strains of Bordetella have
fimbriae. Cocobacilli in the smear are arranged separately, in pairs and short
chains.

2.3.2. Cultural characteristics

B. pertussis is an aerobic coccobacillus. No growth occurs anaerobically.
Optimum temperature of growing is 35°C- 37°C. For isolation of bacterium is
necessary to use complex media. Bordetella is difficult to grow without specialized
media. Because it is inhibited by a variety of substances such as heavy metals,
sulphides, fatty acids. Growth is inhibited by the organism's own waste products so
media containing activated charcoal. The organism will not grow on simple media,
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Chocolate agar or MacConkey agar. The common medium used for isolation of
Bordetella is Bordet-Gengou that contains starch-glycerin-blood agar. This media
contains glycerol as a stabilizer, and often an antibiotic such
as cephalexin or methicillin is added to suppress many Gram positive organisms.
Blood is required not to provide additional nutritive factors but to neutralize
inhibitory substances formed during bacterial growth.
Growth of bacteria is slow. Over 3-5 days of incubation in a humidified
atmosphere at 37°C colonies appear small, convex, and glossy. The colonies have
the appearance of droplets of mercury. Colonies are surrounded by a zone of
hemolysis.

Biochemical reactions. Bordetella is biochemically inactive. It does not

ferment carbohydrates. It does not produce indole, do not have nitrate reductase. It
produces oxidase and catalase also.

Resistance. Bordetella is fragile organism and it is very sensitive to various

agents. Microorganism can be killed by heating at 55°C during 30 minutes. It is
inactivated by sunlight in one hour. Outside the body, B. pertussis in dried droplets
is said to survive for five days on glass, three days on cloths and a few hours on
paper. [1]

Antigenic structure. B. pertussis cells have many antigens, most external

are an agglutinogen and a hemagglutinin. The cell wall contains a heat stable toxin,
the protective antigen and a histamine-sensitizing factor. When the cell wall is
destroyed protoplasm produce a heat-labile toxin and other antigens. There are
several serotypes of B. pertussis that may have epidemiologic significance. [7]
Bacteria of the genus Bordetella contain antigen O somatic, it is common for all
genus. It is thermostable.

2.4. Virulence factors

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 The set of genes expressed by B. pertussis which allow it to invade and
persist inside the host are termed virulence factors. These include adhesines which
facilitate attachment to target host cells and toxins which enable the bacterium to
evade the host immune system. Other virulence factors of B. pertussis include:
- dermonecrotic toxin;
- filamentous hemagglutinin;
- tracheal cytotoxin;
- lipopolysaccharide (LPS);
- tracheal colonization factor (Tcf) and the serum resistance locus [7,1]
Pathogenicity factors initiate the infectious process and have different
function. Bordetela toxin promotes adhesion to respiratory epithelium, sensitization
to histamine, lymphocytosis, increase secretion of insulin, stimulate production of
IgE, block phagocyte activity of leucocyte.
Filamentouse hemagglutinin promotes adhesion to respiratory epithelium,
agglutinates erythrocytes. Pertactin stimulates adhesion to respiratory epithelium
and it is an outer membrane protein antigen present in all strains of B. pertussis.
Pertactin is included in acellular pertussis vaccines. Agglutinogenes the same helps
adhesion to respiratory epithelium.

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 Tracheal toxin causes ciliary stasis and necrosis of respiratory epithelium.
Dermatonecrotic toxin causes vasoconstriction a focal necrosis.
Lipopolysaccharides acts as an endotoxin. Adenylate cyclase block phagocyte
activity of leucocytes, induces apoptosis of macrophages, induces hemolysis.
Fimbria promotes adhesion to respiratory epithelium.

2.4.1. Pathogenesis

B. pertussis is an obligate human parasite but infection can be reproduce

experimentally in several species of animals. Infection with B. pertussis is initiated
by attachment of the organism to the ciliated epithelial cells of the nasopharynx.
Attachment is mediated by surface adhesins (e.g., pertactin and filamentous
hemagglutinin), which bind to the integrin family of cell-surface proteins,
probably in conjunction with pertussis toxin. The role of fimbriae in adhesion and
in maintenance of infection has not been fully delineated. At the site of attachment,
the organism multiplies, producing a variety of other toxins that cause local
mucosal damage (tracheal cytotoxin, dermonecrotic toxin). Impairment of host
defense by B. pertussis is mediated by pertussis toxin and adenylate cyclase toxin.
There is local cellular invasion, with intracellular bacterial persistence; however,
systemic dissemination does not occur. Systemic manifestations (lymphocytosis)
result from the effects of the toxins.
The pathogenesis of the clinical manifestations of pertussis is poorly
understood. It is not known what causes the hallmark paroxysmal cough. A pivotal
role for pertussis toxin has been proposed. Proponents of this position point to the
efficacy of preventing clinical symptoms with a vaccine containing only pertussis
toxoid. Detractors counter that pertussis toxin is not the critical factor because
paroxysmal cough also occurs in patients infected with B. parapertussis, which
does not produce pertussis toxin. It is thought that neurologic events in pertussis,
such as seizures and encephalopathy, are due to hypoxia from coughing paroxysms
or apnea rather than to the effects of specific bacterial products. B. pertussis

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pneumonia, which occurs in up to 10% of infants with pertussis, is usually a
diffuse bilateral primary infection. [12]

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 2.5. Clinical manifestation

Whooping cough tends to develop in stages, with mild symptoms occurring

first, followed by a period of more severe symptoms, before improvement begins.
Clinical manifestation is localized in respiratory tract and does not disseminate.
Paroxysmal cough is not hallmark. Patients show variation in symptoms. If the patient is
younger the disease is more severe. For infection is specific three clinical stages:
- catharal - it is affected upper respiratory tract;
- paroxysmal- infection extends to lower respiratory tract with severe
cough;
- convalescent – less cough and persist several months. [3]

If a person with whooping cough sneezes, laughs, or coughs, small droplets

that contain the bacteria may fly through the air. You might get sick when you
breathe the droplets. When the bacteria get into your airways, they attach to the
tiny hairs in the linings of the lungs. The bacteria cause swelling and
inflammation, which lead to a dry, long-lasting cough and other cold-like
symptoms.

Pertussis is a prolonged coughing illness with clinical manifestations that

vary by age. Classic pertussis is most often seen in preschool and school-age
children. After an incubation period averaging 7—10 days, an illness develops that
is indistinguishable from the common cold and is characterized by coryza,
lacrimation, mild cough, low-grade fever, and malaise.

After 1—2 weeks, this catarrhal phase evolves into paroxysmal phase. The
cough, in this stage becomes more frequent and spasmodic with repetitive
explosions of 5—10 coughs, often within a single expiration. Post tussive
vomiting is frequent, with a mucous plug occasionally expelled at the end of an

21
episode. The episode may be terminated by an audible whoop, which occurs upon
rapid inspiration against a closed glottis at the end of a paroxysm. During a spasm,
there may be impressive neck-vein distension, bulging eyes, tongue protrusion,
and cyanosis. Paroxysms may be precipitated by noise, eating, or physical contact.
Between attacks, the patient's appearance is normal but increasing fatigue is
evident. The frequency of paroxysmal episodes varies widely, from several per
hour to 5—10 per day. Episodes are often worse at night and interfere with sleep.
Weight loss is not uncommon as a result of the illness's interference with eating.
Most complications occur during the paroxysmal stage. Fever is uncommon and
suggests bacterial superinfection.
After 2—4 weeks, the coughing episodes become less frequent and less
severe—changes heralding the onset of the convalescent phase. This phase can
last 1—3 months and is characterized by gradual resolution of coughing episodes.
For 6—12 months, intercalated viral infections may be associated with a
recrudescence of paroxysmal cough. [ 12 ]
The disease lasts 6-8 weeks although in some it may be very prolonged. The
infection is limited to respiratory tract. Bacilli do not invade de blood stream. In
the initial stage the bacilli are limited to the nasopharynx, bronchi and trachea.
Clumps of bacilli can be seen tangled in the cilia of the respiratory epithelium. As
the infection progresses inflammation extend to the lungs, producing diffuse
bronchopneumonia.[1]

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 2.5.1. Complications

Complications are frequently associated with pertussis and are more common
among infants than among older children or adults. There are some risk factors of
complications. Risk factors may be aggravated by:
- prematurity
- anemia
- hypotrophy
- age under 1years(especially under 6 months)
- vomiting more 4 times per day
- absent or incomplete vaccination
- presence of apnea
- chronic pulmonary insufficiency
- chronic cardiac insufficiency
- chronic adrenal insufficiency
- lungs defects
- bronchial asthma
- hypovitaminozis A
- CNS disease

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 Subconjunctival hemorrhages, abdominal and inguinal hernias,
pneumothoraxes, facial and truncal petechiae can result from increased
intrathoracic pressure generated by severe fits of coughing. Weight loss can follow
decreased caloric intake. In contrast to the primary B. pertussis pneumonia that
develops in infants, pneumonia in adolescents and adults with pertussis is usually
caused by a secondary infection with encapsulated organisms such as Streptococcus
pneumoniae or Haemophilus influenzae. Pneumothorax, severe weight loss,
inguinal hernia, rib fracture, carotid artery aneurysm, and cough syncope have all
been reported in adolescents and adults with pertussis.

The worst and tragic complication is death. This is rare except in young
babies for whom it is a more exhausting illness than some can support. In babies it
can lead to respiratory failure, convulsions and coma from encephalopathy. In the
underdeveloped world, the mortality is vastly greater. [23]

2.6. Immunity

Recuperation from pertussis is followed by immunity. The same after

adequate vaccination remain antibodies. Reinfection may occur but are mild.
Severe clinical cases can be observed in adults. It is because the first defense
against B. pertussis infection is the antibody that prevents attachment of the
bacteria to the epithelium of the respiratory tract. In blood are accumulated
antitoxins, agglutinins, precipitins. Cell-mediated immunity and the same humoral
are considered to be important in pertussis. Same years ago was considered that
immunity after natural infection is lifelong. Epidemiological data demonstrate, that
it clearly is not and that additional episodes of clinical pertussis are prevented by
intermittent subclinical infection.

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 2.7. Laboratory diagnosis
Methods of diagnosis of Whooping cough
Early diagnosis of pertussis could limit its spread to other sensitive persons. It
is difficult to diagnose whooping cough in its early stages because the clinical
symptoms resemble those of other common respiratory illnesses, such as the flu,
cold, bronchitis, pneumonia. Doctors can diagnose whooping cough only by asking
about symptoms and listening to the cough. If pertussis is developed with classic
symptoms, to diagnose clinically infection is not difficult. But especially in older
children and adults, it is difficult to differentiate infections caused by B. pertussis
and B. parapertussis from other respiratory tract infections on clinical reasons.
Clinical tests may be necessry, but to confirm infection is indicated
laboratory tests. The following microbiological methods are used in diagnosis of
pertussis:

- microscopical method

- bacteriological method

- modern methods of diagnosis

- serological method

Microscopical, bacteriological and PCR are direct method of diagnosis of B.

Pertussis.

2.7.1.Specimens.

Harvesting and transport of biological specimens is important in the isolation

and identification of causal agents of pertussis. As material for examination are
used sputum nasopharyngeal swabs, nasopharyngeal aspirates. Specimens are
taken from children, adolescents infants and adults. The same is taken blood, but it
is used in serological method of diagnosis.

25
 For bacteriological methodnof diagnosis specimens can be collected by
different methods:

a) The peroral swab: First, it is important to avoid contamination with

saliva. Secretions are collected from posterior pharyngeal wall with a cotton swab
on a bent wire passed through the mouth.

b) The pernasal swab: Swab on a flexible nichrome wire is passed along

the floor of the nasal cavity and material collected from the pharyngeal wall.
Aspirate from nasopharynx collected through catheter.

c) The cough plate method: Culture plate is held about 15cm in front of
pacient’s mouth during coughing . The droplets of exudate directly reach to the
nutrition medium.

Transport of biological specimens. B. pertussis is delicate bacteria. The NPS

or NPA should, after collection be transported quickly to the microbiology
laboratory for culture. Samples are colected and transported at room temperature.

2.7.2. Direct microscpy.

From nasopharyngeal secretions is made smear. Subsequent smear is colored

by Gram method. In smear is observed Gram- negative cocobacteria, arranged
solitary. Bacteria do not have flagella and spores, but are capsulated. Microscopy
as a first step is indicative and it has to be confirmed by culture.

Figure . Under the microscope Bordetella are often bipolar staned and appear
singly or in pairs

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 Source: Copyright © 1995 University of
Texas - Houston Medical School, DPALM MEDIC, All rights reserved.

2.7.3. Bacteriological method of diagnosis

Culture of nasopharyngeal secretions remains the gold standard of diagnosis.

By cultivation can be confirmed up to 80-90% of cases of pertussis. Because B.
pertussis is very sensitive to drying, secretions for culture should be inoculated
without delay onto appropriate medium (Bordet-Gengou or charcoal agar). B.
pertussis is an obligate aerobic bacteria , growing occur in aerobic conditions.

In the first stage is inoculated nasopharyngeal swab or sputum on Bordet

Gengou medium. All plates are incubated for seven days at 35–36 °C and inspected
at day 3 and 7 after inoculation. If typical colonies appear, they are re-isolated and
identified. Plates should be incubated for seven days before being discarded as
negative.

In the second stage is examined the daily appearance of typical colonies. The
same is done macroscopic examination of colonies. Typical B. Pertussis colonies
on Bordet-Gengou plates are small (1 mm in diameter after three days of culture),
like mercury droplets and glistening. On Bordet-Gengou plates, Bordetella appears
haemolytic. At this stage are examined all the cultural property and some discret
colony are transfered on agar slant. It is done for accumulation of bacteria.

In the third stage is identified pure culture according to some properties of of

Bordetella:

27
 a) Microscopic. It is checked purity of the growth by performing a gram
stain. They appear as Gram-negative coccobacilli, non sporing, non motile.

b) Culture character. It is aerobic bacteria. Grows optimally at 35°-37°C.

Bordetella is fastidiouse bacteria and grows on special medium.Preffered medium
is Bordet Gangou. Growth takes longer up to 48-72 hours. Mercury drop colonies
on Bordet Gangou.

c) Biochemical characters. Determine biochemical characters, such as

oxidase, urease, nitrate-reductase or carbohydrate utilization after subculture on
BG or RL agar medium.

d) Antigenic identification. Perform slide agglutination with antibodies

to B. pertussis and B. Parapertussis. Identification of antigen structure of bacteria
is done after isolation. It is used specific serum for agglutination or
immunofluorescence tests.

In the fourth stage is analyzed the results of identification tests. Analysis

report is drawn. Confirm if it was isolated Bordetella.

2.7.4. Modern techniques of cultivation of Bordetella

a) BACTEC system

Traditional cultivation techniques used for diagnosis of pertussis require

more time and qualified staff. For these reasons they were invented more efficient
and more effective methods in diagnosis of infection. Automated systems have
been developed for cultivation and detection of bacterial growth. These systems
can quickly and accurately detect bacteria in specimen. Example of modern
method is BACTEC system. BACTEC system is based on detection and
measurement of carbon dioxide (CO2) produced by microorganisms.

b) RT-PCR (Real-time polymerase chain reaction)

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 The polymerase chain reaction is based on a technology in vitro. It consists in
a rapid generation of a nucleotide sequence of a pathogen. The PCR amplification
product is then detected by various methods.

Real-time polymerase chain reaction (RT-PCR) is expensive, but more

sensitive and more rapid than culture. RT-PCR may be used as an alternative for a
rapid diagnosis of whooping cough, and it is used in addition to, and not istead of
culture. PCR is a rapid test with greater sensitivity than culture (70 to 99%), but
less specificity (86-100%). Some PCR tests do not differentiate between closely
related species of Bordetella. Organisms do not need to be viable, and the test may
be positive post-antibiotics.

2.7.5. Serological method of diagnosis.

Serological method is used for confirmation of diagnosis and for

determination of antibodies in serum. Antibodies againstBordetella appear late as
2-3 weeks. Antibody titers increased gradually until 8-10 weeks. Two serum
samples are collected with interval of 3 weeks. Because cough syndrome is
polyetiologic in the beginning is done complement fixation test. The reaction is
performed with several types of B.pertussis antigens, B. parapertussis,
parainfluenza virus, adenovirus. Significant is the increase 4-fold titer antibodies
against antigens possibile.

Antibodies to Bordetella can be detected by the following types of reactions:

- agglutination test;

- indirect hemagglutination test;

- ELISA test;

- immunofluorescence reaction.

Antibodies to pertusis toxin, to peractin, to hemagglutinin and against fimbria

can be determined. Determining of IgM immunoglobulin indicates an acute

29
infection, and if it is present IgG indicate chronic infection. Serological tests on
patients are of lttle diagnostic help because a rise in agglutinating or precipitating
antibodies does not occur until the third week of illness.

2.7.6. Differential diagnosis

A child presenting with paroxysmal cough, post tussive vomiting, and whoop
is possible to be suspected with B. pertussis or B. parapertussis. Increasing
numbers of lymphocytes indicate infection with B. Pertussis. Adults and
adolescents that do not have whoop or another specific clinical symptoms as cough
the differential diagnosis of a prolonged coughing is more extensive. Viruses such
as influenza, respiratory syncytial virus and adenovirus have been isolated from
patients with clinical pertussis but probably represent co-infection. Whooping
cough has to be suspected when any patient has a cough that does not improve
within 14 days. Other agents have to be taken in consideration include infections
caused by Mycoplasma pneumoniae, Chlamydia pneumoniae, adenovirus, influenza
virus, and other respiratory viruses.

2.8. Prevention “Vaccinate, vaccinate, vaccinate.”

For adults, children and adolescents whooping pertussis, is a huge problem.

Infection is started with cold symptoms, followed by a cough that takes weeks or
months to resolve. Lost work and school are common. For infants who haven not
yet been immunized, pertussis can be severe even life threatening. Children
usually have severe illness requiring hospitalization and are at high risk for
complications like pneumonia and seizures.
Preventing whooping cough starts by recognizing how young children usually
catch the bacteria: from other family members. In most cases, it’s a parent or
sibling that transmits pertussis to a child. Bordetella is a bacterium that can live in
the human respiratory tract. The bacteria is easily spread through sneezes
and coughs, often from people who often don’t even know they have the infection.
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Pertussis vaccine immunity is short lived. The whooping cough vaccine, like
natural pertussis infection, does not provide lifelong protection. Antibodies to
pertussis disappear during 10 years after the last childhood vaccine, letting
adolescents and adults sensitive to infection. People who have had pertussis lose
their immunity, too.
The best way to prevent whooping cough is with the pertussis vaccine,
which doctors often give in combination with vaccines against two other serious
diseases — diphtheria and tetanus. Doctors recommend beginning vaccination
during infancy.

The vaccine consists of a series of five injections, typically given to children

at these ages:

 2 months
 4 months
 6 months
 15 to 18 months
 4 to 6 years

After the third dose, children are well protected. They have about 80% to 85%
immunity to pertussis. If they do catch whooping cough despite the vaccine, the
infection is usually mild. But in their first six months and especially the first two
months of life before babies have been vaccinated they are particularly vulnerable
to serious whooping cough infections. Because of this, for infants with pertussis
who are less than two months old, severe illness is specific. Infants require
hospitalization, because of complication develop a pneumonia, and can die because
of whooping cough.

Preventing transmission to all children and especially to infants, is the major

public health issue. The first and most important rule of pertussis prevention is not
complicated. “Vaccinate, vaccinate, vaccinate.” Vaccination is the single best way
to prevent whooping cough.
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 2.8.1. Pertussis vaccine

Pertussis vaccine is a vaccine that protects against whooping cough. The first
vaccine against pertussis was developed in the 1930s by Leila Denmark. It
included whole-cell killed Bordetella pertussis bacteria. There are two types:
whole-cell vaccines and acellular vaccines. The vaccine is estimated to have saved
over half a million lives in 2002. The acellular vaccine is more commonly used in
the developed world due to less side effects. The whole-cell vaccines
develop complications. New acellular pertussis vaccines were developed in the
1980s, which included only a few selected pertussis antigens (toxins and adhesins).

Side effects include the following: Local reactions, such as fever, redness and
swelling at the injection site, and soreness and tenderness where the shot was
given, are not uncommon in children and adults.

Other indications to prevent pertussis, or nonspecific prevention.

Vaccination is a specific method of prophylaxis. Vaccination must be carried out
according to immunization plane. Other than vaccination and booster
immunization with Tdap, there’s no effective way to prevent pertussis. B. pertussis
is too contagious, and the symptoms too similar to those of the common cold, to
realistically stop its spread. Still, there are two things you can do to reduce the
symptoms and spread of whooping cough:

Active detection and early isolation of patient at home or in infective

hospital for a period of 25 days after onset disease
The mandatory registration of case disease.
Patient treatment will last in light and medium forms 1-2 weeks, and in the
severe or complications 2-3 more weeks.
Detection and monitoring of contact persons time of 14 days from the last
event.
Clinical examination is done by the family doctor.

32
 Quarantine measures are included in the collective of children for 14 days
from the last case.
It is necessary to do ventilation of rooms and wet cleaning 2-3 times per day.
It is necessary to wash hands. Hygiene of hands is a universal
recommendation. When it is possible, wash hands or use alcohol-based rubs after
touching nasal secretions.
Cover your nose and mouth when coughing or sneezing. Encourage children
to do the same.

33
 2.9. Treatment of whooping cough

Pertussis is generally treated with antibiotics and early treatment is very

important. The goal of antibiotic therapy for whooping cough is to remove the
bacteria from the nasopharynx. Adequate treatment may make infection less
serious if it is started early, before coughing fits begin. Treatment can also help
prevent spreading the disease to close contacts.
Obligatory recommendations. The regime of day has to be respected.
Patients will be left as much time in open air. Temperature and humidity to be
reasonable. It frees the airways by postural drainage and aspiration of secretions.
The same it has be respected diet. Food must contain necessary ingredients rich in
vitamins and microelements. Food should be easily digestible. Foods does not
have to stagnate in the stomach, small meals will be prescribed repeated. Ensure
adequate hydration (mineral water, juices, compote, teas.

Antimicrobial Choice
Macrolide antibiotics are the drugs of choice for treatment of pertussis.
Macrolide-resistant B. pertussis strains have been reported but are rare. The
recommended antimicrobial agents for treatment or chemoprophylaxis of
pertussis are azithromycin, clarithromycin, and erythromycin. Trimethoprim-
sulfamethoxasole can also be used. Trimethoprim-sulfamethoxazole is
recommended as an alternative for individuals allergic to macrolides.
The choice of antimicrobial should be made after consideration of the:
 Cost
 Ease of adherence to the regimen prescribed
 Potential for adverse events and drug interactions
 Tolerability
Besides antibacterial therapy is indicated symptomatic treatment. Also ensure
and supportive care for patients. Young infants have the highest rates of
complication and death from pertussis; therefore, most infants should be
34
hospitalized. A quiet environment may decrease the stimulation that can trigger
paroxysmal episodes.
Apart from antibiotics administered cough suppressants. To drain or
liquefaction sputum is indicate expectorant. Mucolytics helps to eliminate bacteria
from the respiratory tract.
At the necessity will be given antihistamines. After antibiotic treatment to
restore normal flora will be administered probiotics.
Hyperimmune globulin is prepared from sera of immune person repeatedly
injected with pertussis vaccine. Sera can be given to debilitated or unimmunized
children very early in the illness with possible benefit.

35
 III. MATHERIALS AND METHODS OF RESEARCH
This paper is an analytical descriptive study of the diagnosis of whooping
cough. For this purpose the method used was a literature review. The information
sources are publications, articles, and also books in mainly in English and some in
Romanian as well, including from internes sources.
The goal of this thesis is to review the existing information on the diagnosis
of whooping cough and to analyze the methods that are used for this purpose. It
compares make the advantages and disadvantages for each of these methods.
Although this research was not meant to conduct a practical study, nor to test new
diagnostic methods, it describes the aspects related to prophylaxis and treatment of
pertussis. And even if they are not the object of this research, they are included as
well because they can point out the importance of timely diagnosis. This is relevant
because currently, based on the data analyzed, the finding is that this is a long-
lasting disease.
The aspects studies include the historical evolution of this infection together
with its virulence factors. The source of infection and way of transmission were
also studied and the clinical symptoms of the disease are also described.
The following diagnosis methods described and analyzed:
- the clinical method;
- the microscopic method;
- the bacteriologic method;
- the serologic method;
- the polymerized chain reaction method (PCR);

Currently these represent all the methods used both at national level as well as
at international level. They cover both the classic methods and the more
contemporary ones such as PCR or ELISA, even if some of them are, for various
reasons, used less in the laboratories from Moldova. This paper also analyzes the
factors that contribute to some of the methods being preferred to the disadvantage
of the others.

36
 For each of these diagnosis methods the following research methods were
used:
- theoretical analysis;
- theoretical synthesis;
- deduction;
- the historical-bibliographical study;
- the statistical-mathematical method

This research also presents a statistical analysis of the data on incidence of

morbidity and probability of mortality in connection with this infection. These data
were taken from studies conducted by national organizations and institutions such
as the National Bureau of Statistics and international ones such as the world Health
Organization (WHO) and the reports they published.

37
 IV. RESULTS OF THE STUDY
As a result of this study we now have an overview of the existing information
on the entire process from the moment of infection, its development and classic
and modern methods of diagnosis of whooping cough, as well as aspects of
prevention and treatment.
The diagnosis is conducted through 4 main methods;
- the microscopic method;
- the bacteriologic method;
- the serologic method, and
- modern methods (PCR, ELISA, BACTEC).
This research also gathered and analyzed statistical data from international
organizations such as World Health Organization for a period since 1990 and for
the current years 2010-2014, covering several European countries including
Moldova and countries from other regions in the world.
It also presents a statistical review from the data of the National Center for
Public Health of Moldova for 2012-2014 as well.

38
 V. CONCLUSIONS
From the material presented in the chapters of this paper we find specific
information about the causal agent isolated and identified from the specimen and
the assessed susceptibility to antibiotics.
The findings refer to several different aspects as follows.
This paper also analyzed the historical evolution of the infection and its
treatment and prophylaxis, due to the fact that early on there was no antibiotic
treatment, the infection was often deadly or had sequelae. And starting with the
period between 1945-1960, when the pertussis vaccination was introduces, a
considerable improvement has been observed.
From the diagnosis methods mentioned in the respective chapter, the finding
of this theoretical review is that the most efficient is considered to be the
bacteriological method. This is because it is a direct method and it can determine
also the sensitivity to antibiotics. Due to this fact, an elective antibiotic can be
chosen. This significantly simplifies the treatment, and the possible complications
compared to the situation when the infection would be left untreated for a longer
period until another diagnosis method is used.
The other modern methods are also very efficient, a good example is the PCR
method, but at the same time it is less used due to higher costs. For the same
reason it is not yet used in Moldova.
The serological method is becoming less and less important because
whooping cough is a long-lasting disease and it provides insufficient information
compared to the bacteriological method.
Currently according to the existing data mentioned in the statistics section of
this research, an increase of the case of whooping cough is observed. This fact is
based on two possibilities. The first factor is that in the current modern society
people are more informed and awareness is higher, hence they can better
discriminate between the symptoms of whooping cough and regular colds or flues,
as well as are more inclined to see a medical specialist sooner than in the previous
years. The second possibility is connected to the current trend to refuse vaccination
39
because of the alleged complications and possible cases of autism in children as a
result of the pertussis vaccine.

This increase is noticed worldwide, in countries such as the USA, European

countries, the Australian region and also in Moldova as well.

Due to early diagnosis, the treatment has also improved, currently the most
efficient treatment includes macrolides such as erythromycin, etc.

From the perspective of prophylaxis we also conclude that vaccination has an

increased role in preventing the spread of this infection, as it is seen from the
statistical data for the period since it was introduced. The important factor here is
that prophylaxis is done according to an immunization plan with acellular vaccine.

The main conclusion is that this infection, although dangerous if not

diagnosed and treated, is not a hopeless incurable disease. And with proper
awareness and population education it can be controlled through proper prevention,
timely diagnosis and appropriate treated.

40
 V. BIBLIOGRAPHY AND REFERENCES

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