CHAPTER I
1 INTRODUCTION
Respiratory system can be divided into upper and lower tracts. The respiratory and gastrointestinal tracts are the two major connections between the interior of the body and the outside environment. The respiratory tract is the pathway through which the body acquires fresh oxygen and removes unneeded carbon dioxide. It begins with the nasal and oral passages (Forbes et al., 2002).
The upper respiratory tract is frequently the site of general and localized infections. It is the primary site of infection for most viral diseases, which are spread by sneezing, coughing or direct contact with materials contaminated by respiratory secretions. Although the majority of such symptoms are viral in origin, secondary bacterial infection may often follow, particularly in the very young and malnourished. Resident bacteria in the upper respiratory tract such as Haemophilus influenzae, Streptococcus pneumoniae and Streptococcus pyogenes are the most common causes (Greenwood et al., 2003).
Respiratory tract infection is the major health problem in developing countries. Infection of the respiratory tract is the most frequent and important cause of short term illness in the population. It is frequently the first infection to occur after birth, and too often the final illness before death (especially pneumonia) (Dawadi et al., 2005).
The respiratory tract is the most common site for infection by pathogens. This site becomes infected frequently because it comes into direct contact with the physical environment and is exposed to the microorganisms in the air (Chantler and Griffith, 2004).
Acute respiratory tract infections (ARIs) are one of the most important causes of morbidity and mortality in children throughout the world. More than 4 million children under 5 years of age are estimated to die from ARI every year. This represents about 30 % of 14.25 million deaths of children under 5 years of age that occur in the developing world each year (Teixeira, 2002).
ARIs are estimated to be responsible for one third of all childhood deaths in developing countries. It is estimated that Bangladesh, India, Indonesia and Nepal together account for 40% of global acute respiratory infection mortality. These respiratory infections can manifest in any area of the respiratory tract, including the nose, middle ear, throat, voice box, air passage and lungs. As an infection of lungs, pneumonia is one of the major causes of ARI. About 90% of ARI deaths are due to pneumonia, which usually is bacterial in origin (WHO, 2000).
Approximately 2.6 million children under 5 years of age die annually of pneumonia predominantly in the developing world; approximately one half of these deaths are attributable to S pneumoniae either solely or in conjunction with a viral respiratory infection, malnutrition or HIV infection (O’brien et al., 2003).
Streptococcus pneumoniae are involved chiefly in the infections of upper and lower respiratory tracts. The pneumococci, in low numbers, is a part of the normal nasopharyngeal and oropharyngeal flora of many healthy persons and also children, which generally remains harmless unless it is provoked by a viral infections such as influenzae or the common cold to spread to the lower respiratory tract, middle ear, paranasal sinus or the blood. In this situation, pneumococci are secondary pathogen but may be primary pathogen in immunocompromised people.
Bacterial colonization of nasopharynx starts immediately after birth and continues throughout life with small changes. However, a major part of infections is caused by microbes like S pneumoniae, H influenzae, M catarrhalis, N meningitidis and Staphylococcus aureus, which originally belong to normal flora (Kaijalainen, 2006). Colonization frequently occurs without the development of disease (Catterall, 1999).
People usually carry pneumococci without symptoms, but carriage can also contribute to respiratory or even systemic disease. Several factors have considerable impact on pneumococcal carriage and its rates (Kaijalainen, 2006).
The bacteria S pneumoniae carried in the nasopharynx of children reflect the infection causing strains currently circulating in the community. So studies of the prevalence of different pathogens and their resistance patterns can provide useful indications for more rational therapeutic and preventive strategies. The symptomatic nasopharyngeal carriage of S pneumoniae is widely prevalent in young children and has been related to the development of disease and the spread of the pathogen. Furthermore, nasopharyngeal colonization by antibiotic resistant S pneumoniae has steadily increased over the last few years. Antibiotic resistant strains are more often carried by infants and young children than adults and belong to a limited number of serotypes that are also some of the most common cause of invasive pediatric diseases (Marchisio et al., 2002).
There was a belief among some commentators a generation ago that infectious disease was a problem that was well on the way to permanent resolution owing to the development of effective vaccines and antibiotics. However, such complacency has now completely disappeared (Denyer et al., 2005) and infectious diseases had still remained the leading cause of death all over the world.
Considering the above mentioned facts, this dissertation work as the partial fulfillment for Master Degree was conducted among the children attending Kanti Children’s Hospital in order to determine the prevalence of nasopharyngeal carriage of S pneumoniae, its antibiotic susceptibility pattern and distribution of its serotypes.
CHAPTER II
2 Objectives
This study was conducted with the following objectives
2.1 General objective
To assess sero-epiemiology of Streptococcus pneumoniae and determine its antibiotic susceptibility pattern in children less than 5 years of age attending out patient department of Kanti Children’s Hospital.
2.2 Specific objectives
1. To determine the prevalence of nasopharyngeal carriage of Streptococcus pneumoniae among children attending Kanti Children’s Hospital.
2. To isolate and perform antibiotic susceptibility test of Streptococcus pneumoniae from nasopharyngeal swab.
3. To serotype the isolates employing co-agglutination method.
4. To perform MIC for Oxacillin resistant strains.
CHAPTER III
3 Literature review
3.1 Respiratory tract infection
Respiratory tract infection is a major health problem in developing countries. Infection of the respiratory tract is the most frequent and important cause of short term illness in the population. It is frequently the first infection to occur after birth, and too often the final illness before death (especially pneumonia) (Dawadi et al., 2005).
Respiratory tract infections occur more frequently than they are reported and are often thought of as inconveniences of life that will pass away quickly (Dawadi et al., 2005). Respiratory tract infection, comprising a broad spectrum of diseases from self limiting acute bronchitis to severe pneumonia, is caused by a wide range of microbial pathogens (Hosker, 1994).
The human respiratory tract is exposed to many potential pathogens via the smoke, soot, and dust that are inhaled from the air. It has been calculated that the average individual ingests about 8 microorganisms per minute or 10,000 per day (WHO, 2003).
Children are smaller; their surface area is greater in comparison to the body weight therefore they tend to have higher metabolic rate and due to these reasons they have higher respiratory rate than the adults. So, this means they ingest more microorganisms than adult (www.medscape.com).
The respiratory tract is the most common site for infection by pathogens. This site becomes infected frequently because it comes into direct contact with the physical environment and is exposed to the microorganisms in the air (Chantler and Griffith, 2004).
3.2 Organisms present in the nasopharynx and oropharynx of healthy human
Possible pathogens Rarely pathogens
Acinetobacter spp
Viridans streptococci
Beta hemolytic streptococci
Streptococcus pneumoniae
Staphylococcus aureus
Neisseria meningitidis
Mycoplasma spp
Haemophilus influenzae
Haemophilus parainfluenzae
Moraxella (Branhamella) catarrhalis
Candida albicans
Herpes simplex virus
Enterobacteriaceae
Mycobacterium spp
Pseudomonas spp
Klebsiella ozaenae
Bacteroides spp
Peptostreptococcus spp
Actinomyces spp
Haemophilus aphrophilus
Entamoeba gingivalis Nonhemolytic streptococci
Staphylococci
Micrococci
Corynebacterium spp
Coagulase negative staphylococci
Lactobacillus spp
Veillonella spp
Spirochetes
Campylobacter spp
Source: Forbes et al, 2002
3.3 Epidemiology of ARI among children
Acute respiratory tract infections (ARIs) are one of the most important causes of morbidity and mortality in children throughout the world. More than 4 million children under 5 years of age are estimated to die from ARI every year. This represents about 30 % of 14.25 million deaths of children under 5 years of age that occur in the developing world each year (Teixeira, 2002).
According to the WHO Report 2000, the top five respiratory diseases account for 17.4% of all deaths and 13.3% of all Disability Adjusted Life Years (DALYs). Also, out of total acute respiratory disease, 20-24% of deaths are accounted for by lower respiratory tract infection (Verma, 1981).
Lower respiratory tract infections are the major burden of premature death and disability worldwide and as might be predicted, the burden is significantly greater in the developing countries compared with the developed world (Cant et al., 2002).
3.4 Respiratory tract infections in developing world
ARIs are estimated to be responsible for one third of all childhood deaths in developing countries. Although the incidence of ARI, at 5-9 episodes/child/year in the first 5 years of life, is the same in developed and developing countries, the incidence of acute lower respiratory tract infection (ALRI) is over 12- fold greater in developing countries (Cant et al., 2002).
Risk factors for progression from ARI to ALRI include young age (0-11 months), malnutrition (both macro- and micro- nutrients), lack of breast feeding, HIV infection and environmental factors such as crowding and indoor air pollution (Cant et al., 2002).
It is estimated that Bangladesh, India, Indonesia and Nepal together account for 40% of global acute respiratory infection mortality. These respiratory infections can manifest in any area of the respiratory tract, including the nose, middle ear, throat, voice box, air passage and lungs. As an infection of lungs, pneumonia is one of the major causes of ARI (WHO, 2000).
3.5 Causative agents of RTIs
It is clear that bacteria figure prominently in both primary and secondary roles in acute upper and lower respiratory tract infections. Many of the bacteria that cause ARI can be isolated as a part of the normal flora of healthy people. Under certain circumstances, these colonizing microorganisms go on to cause disease (Teixeira, 2002).
Bacterial infections of the respiratory tract can be grouped according to their symptomatology and anatomic involvement. Some of the causative agents are associated with specific syndromes (Teixeira, 2002).
Acute upper respiratory infections are usually benign, transitory and self limited, although some exceptions such as epiglottitis and laryngotracheitis may be severe diseases in small children and neonates. Most of the severe bacterial epiglottitis cases are caused by Haemophilus influenzae. Other severe bacterial infections of the upper respiratory tract are whooping cough (pertussis) caused by Bordetella pertusis and diphtheria caused by Corynebacterium diphtheriae. Pharyngitis, one of the most common bacterial infections, especially in the pediatric age group, is most often caused by Streptococcus pyogenes. H influenzae and S pneumoniae account for the vast majority of sinusitis cases. The most common bacterial pathogens recovered from middle ear of children with acute otitis media are S pneumoniae, H influenzae and Branhamella catarrhalis (Teixeira, 2002).
Pneumonia is the main lower respiratory tract infection, with characteristics much more severe than most of the upper ARI. About 90% of ARI deaths are due to pneumonia, which usually is bacterial in origin (WHO, 2000).
3.6 Diseases caused by Streptococcus pneumoniae
Serious pneumococcal infections occur throughout life, but young children under 5 years old (especially among those under 2 years old) and the elderly are at the highest risk for severe pneumococcal disease. Diseases caused by S pneumoniae include pneumonia (usually lobar type), paranasal sinusitis, otitis media, meningitis, bacteremia, conjunctivitis, etc. Furthermore, more than 90% of pneumococcal pneumonia deaths in children occur in developing countries and pneumococcal meningitis kills or disables over 40% of the children who get the disease (www.infectioncontroltoday.com).
Approximately 2.6 million children under 5 years of age die annually of pneumonia predominantly in the developing world; approximately one half of these deaths are attributable to S pneumoniae either solely or in conjunction with a viral respiratory infection, malnutrition or HIV infection (O’brien et al., 2003).
S pneumoniae continues to be a leading cause of pneumonia, meningitis and otitis media in persons of all ages. Pneumococci are also the most frequent cause of otitis media and bacteraemia and an important agent of sinusitis in children. There is evidence suggesting that most children experience some kind of pneumococcal infection. Approximately 80% of all children experience at least one attack of otitis media by the age of 3 years and pneumococci account for about half of these cases (Teixeira, 2002).
Streptococcus pneumoniae was identified as a major respiratory pathogen shortly after its isolation in 1881. Despite a century of intensive study and antibiotics which readily kill the organism, respiratory tract infections caused by the S pneumoniae remain a formidable problem. S pneumoniae is the commonest cause of community acquired pneumonia (Catterall, 1999).
3.6.1 Mode of transmission
Pneumococci are a part of the normal microbial flora of the nose and pharynx, particularly among young children and are easily transmitted. Infection usually occurs in a person already colonized with S pneumoniae, when the bacteria invade into the patient’s body. Person to person spread is rare, however S pneumoniae can be spread from person to person by inhalation of respiratory droplets (e.g., coughing, sneezing) from an infected person, by direct contact and indirectly by contact with articles such as clothing or tissues freshly soiled with respiratory secretions (Streptococcus pneumoniae, 2004).
Also, the transmission of pneumococci is increased during the course of other respiratory infections when secretions, coughing and sneezing are increased. Although transient nasopharyngeal colonization rather than disease is the normal outcome of exposure to pneumococci, bacterial infection following penetration of the mucosal layer, may occur in persons susceptible to the involved serotype (WHO, 2006).
3.6.2 Incubation period
Due to the fact that many infections arise from bacteria the patient is already carrying it is hard to identify an incubation period, however if a person develops an infection from a new exposure it is commonly within 1-3 days. It is also dependent upon how the illness manifests (Streptococcus pneumoniae, 2004).
3.6.3 Reservoir
S pneumoniae is commonly found in the upper respiratory tract of healthy people as a part of normal flora (Streptococcus pneumoniae, 2004).
3.7 Pneumococcus as a part of normal nasopharyngeal flora
Bacterial colonization of nasopharynx starts immediately after birth and continues throughout life with small changes. Normal nasopharyngeal bacterial flora develops during the first year of life and the number of bacterial species varies much. Normal flora has an important role in the prevention of infectious diseases. However, a major part of infections is caused by microbes like S pneumoniae, H influenzae, M catarrhalis, N meningitidis and Staphylococcus aureus, which originally belong to normal flora (Kaijalainen, 2006).
Pneumococcus has genetic properties allowing it to co-exist with other bacteria and inhibit competing intruders, and by producing hydrogen peroxide, it inhibits the growth of other bacteria such as H influenzae, M catarrhalis and N meningitidis (Kaijalainen, 2006).
3.7.1 Pneumococcal carriage rate
Nasopharyngeal colonization frequently occurs without the development of disease. Colonization can occur within hours of birth and by the 12th postnatal day, the carrier rate is similar to that of the babies’ mothers. Carriage rates are highest in preschool children, children attending child day care centers and nurseries while rates amongst adults depend on the likelihood of contact with other children (Catterall, 1999).
Recent studies during two outbreaks of pneumococcal pneumonia have shown that asymptomatic nasopharyngeal colonization with S pneumoniae frequently results in the production of circulating type specific antibody at levels which confer protection from pneumonia against that serotype. It therefore appears that, although aspiration of colonizing organisms during the first few weeks of colonization may lead to pneumonia, after that time most healthy adults are likely to be protected (Catterall, 1999). Invasive disease is most likely to occur soon after nasopharyngeal colonization with a newly acquired serotype rather than after long duration of carriage of that serotype (O’brien et al., 2003).
The rate of colonization appears to be seasonal, with an increased prevalence seen during the winter (Weber and Rutala, 2003).
Pneumococcus carriage develops among children more rapidly in developing countries compared with industrialized countries (O’brien et al., 2003).
3.7.2 Impacts on pneumococcal carriage rate
People usually carry pneumococci without symptoms, but carriage can also contribute to respiratory or even systemic disease. Several factors, such as age, geographical sites, socio-economic status, family size, indoor air pollution, number of siblings, day care, the presence of upper respiratory tract infection and overcrowded living conditions, have considerable impact on pneumococcal carriage and its rates (Kaijalainen, 2006).
A comparison of pneumococcal carriage rates between studies is difficult due to the variable methodological factors, such as the number and frequency of specimen collection, the quality of specimens and culture techniques. Generally, pneumococcal carriage is highest at the age of 2 years and it decreases over the years (Kaijalainen, 2006).
3.8 Bacterial determinants of virulence
3.8.1 Capsule
A capsule composed of polysaccharide completely envelops the pneumococcal cells. During invasion the capsule is an essential determinant of virulence. The bacterial capsule interferes with phagocytosis by preventing C3b opsonization of the bacterial cells i.e., by interference with binding of complement C3b to the cell surface (Todar, 2003).
The polysaccharide is non toxic and non inflammatory, and the capsule does not appear to engage any host defenses except for the induction of antibody mediated immunity. The pneumococcal capsule is not an antigenic disguise, and it does not impede the activities of underlying components, such as the cell wall and surface proteins, to engage the host defense systems. However, C reactive protein or antibodies to teichoic acid, both of which bind to the cell wall under the capsule, fail to opsonize encapsulated pneumococci (Todar, 2003).
90 different capsular types of pneumococci have been identified and form the basis of antigenic serotyping of the organism. Anti pneumococcal vaccines are based on formulations of various capsular (polysaccharide) antigens derived form the highly prevalent strains (Todar, 2003).
3.8.2 Cell wall (components)
The cell wall of S pneumoniae is roughly six layers thick and is composed of peptidoglycan with teichoic acid attached to approximately every third N-acetylmuramic acid. Lipoteichoic acid is chemically identical to the teichoic acid but is attached to the cell membrane by a lipid moiety. Both the teichoic acid and the lipoteichoic acid contain phosphorylcholine; two choline residues may be covalently added to each carbohydrate repeat. This is an essential element in the biology of S pneumoniae since the choline specifically adheres to choline binding receptors that are located on virtually all human cells (Todar, 2003).
The pneumococcal cell wall is a collection of potent inflammatory stimuli. Challenge with cell wall components alone can recreate many of the symptoms of pneumonia, otitis media and meningitis in experimental models. The phosphorylcholine decorating the teichoic acid and the lipoteichoic acid is a key molecule enabling invasion, and acts both as an adhesion and as a docking site for the choline binding proteins (CBPs). Other respiratory pathogens such as Haemophilus, Pseudomonas, Neisseria and Mycoplasma also have phosphorylcholine on lipopolysaccharide, proteins or fimbriae, suggesting a shared mechanism for invasion of the respiratory tract. Two host derived elements that recognize choline are platelet activating factor (PAF) receptor and the C reactive protein (Todar, 2003).
The peptidoglycan/teichoic acid complex of the pneumococcus is highly inflammatory. Smaller components of peptidoglycan progressively lose specific inflammatory activity. The cell wall directly activates the alternative pathway of the complement cascade, generating chemotaxins for leukocytes, and the coagulation cascade, which promotes a procoagulant state favoring thrombosis. In addition, peptidoglycan binds to CD14, a cell surface receptor known to initiate the inflammatory response for endotoxin. This induces a cytokine cascade resulting in production of interleukin- 1, -6 and tumor necrosis factor from human cells (Todar, 2003).
3.8.3 Surface proteins
On the basis of functional genomic analysis, it is estimated that the pneumococcus contains more than 500 surface proteins. Some are membrane associated lipoproteins, and others are physically associated with the cell wall. The latter includes five penicillin binding proteins (PBPs), two neuramanidases, and an IgA protease. A unique group of proteins on the pneumococcal surface is the family of choline binding proteins (CBPs) (Todar, 2003, Cattrerall, 1999).
Choline binding proteins
Twelve CBPs are noncovalently bound to the choline moiety of the cell wall and are used to “snap” various different functional elements onto the bacterial surface. The CBPs all share a common C terminal choline binding domain while the N-termini of the CBPs are distinct, indicating their functions are different. The CBP family includes such important determinants as PspA (protective antigen), Lyt A, B and C (three autolysins), and CbpA (adhesin) (Todar, 2003).
PspA (pneumococcal surface protein A)
PspA is a protective antigen with 10 choline binding repeats. PspA appears to inhibit complement mediated opsonization of pneumococci, and mutants lacking PspA have reduced virulence.
Autolysin LytA
It is responsible for pneumococcal lysis in stationary phase as well as in the presence of antibiotics. The protein has two functional domains: a C terminal domain with 6 choline binding repeats that anchor the protein on the cell wall, and a N terminal domain that provides amidase activity.
Autolysin LytB is a glucosaminidase involved in cell separation.
Autolysin LytC exhibits lysozyme like activity.
CbpA
It is a major pneumococcal adhesin. It has 8 choline binding repeats. The adhesin interacts with carbohydrates on the pulmonary epithelial surface carbohydrates. CbpA deficient mutants are defective in colonization of the nasopharynx and fail to bind to various human cells in vitro. CbpA also has been reported to bind secretory IgA and complement component C3.
Schematic figure of the known virulence factors of Streptococcus pneumoniae including their main functions and cellular location. Only factors with proven virulence are included
Source: Catterall, 1999
3.8.4 Hemolysins
In addition to surface associated virulence determinants, pneumococci secrete exotoxins. Two hemolysins have been described, the most potent of which is pneumolysin.
Pneumolysin is stored intracellularly and is released upon lysis of pneumococci by autolysin. Pneumolysin binds to cholesterol and thus can indiscriminately bind to all cells without restriction to a receptor. This protein assembles into oligomers to form transmembrane pores which ultimately lead to cell lysis. Pneumolysin can also stimulate the production of inflammatory cytokines, inhibit beating of the epithelial cell cilia, inhibit lymphocyte proliferation, decrease the bacterial activity of neutrophils, and activate complement.
A second hemolysin activity has been described but has not been identified. In addition, pneumococci also produce hydrogen peroxide in amounts greater than human leukocytes produce. This small molecule is also a potent hemolysin.
3.9 Pathogenesis (Transition from colonization to pneumonia and other invasive diseases)
Although S pneumoniae exists in encapsulated and unencapsulated forms, only encapsulated strains have been isolated from clinical material. The importance of the capsule in pneumococcal virulence has been established. However, the capsule itself is not toxic. Composed of one of 90 serologically distinct polysaccharides, the virulence of the capsule lies mainly in its antiphagocytic properties. The level of virulence is determined more by the chemical nature of the capsule then by its size (Catterall, 1999).
The factors which permit pneumococci to spread beyond the nasopharynx are poorly characterized and are likely to vary depending on the virulence of the organism, the state of the host’s defences, and the existence of preceding viral infection. Spread to the lungs probably occurs by aspiration which is aided by impairment of the cough reflex, by increased production of mucus (in which pneumococci also replicate), and by impairment of the mucocilliary escalator. Whilst all of these can be caused by host related disorders, the pneumococcus itself can contribute by pneumolysin dependent disruption of the epithelial type junctions which are essential for the production of mucus. Both influenza virus and adenovirus enhance in vitro adherence of S pneumoniae to respiratory tract epithelial cells (Catterall, 1999).
3.9.1 Colonization
Pneumococci adhere tightly to the nasopharyngeal epithelium through a variety of mechanisms involving the specific interaction between bacterial surface adhesions and epithelial cell receptors. Infection results when colonizing bacteria invade tissue and escape phagocytic defense mechanisms. This commonly occurs when bacteria are transported into the eustachian tube, sinuses, or bronchi (Weber and Rutala, 2003). Passage of pneumococci up the eustachian tube is accompanied by bacterial induced changes in the surface receptors of the epithelial cell, particularly by neuraminidase. Inflammation in the middle ear is caused by pneumococcal cell wall components, and pneumolysin inflicts major cytotoxicity on ciliated cells of the cochlea (Todar, 2003).
Upon reaching the lower respiratory tract by aerosol, pneumococci bypass the ciliated upper respiratory epithelial cells unless there is damage to the epithelium. Instead, they progress to the alveolus and associate with specific alveolar cells which produce a choline containing surfactant (Todar, 2003).
Experimentally, in healthy tissues, it requires approximately 100,000 bacteria/ml to trigger an inflammatory response. However, if a pro inflammatory signal is supplied, inflammation ensues with as few as 10 bacteria. This signal is a cytokine in experimental systems or an intercurrent viral infection in clinical situations. The inflammatory response can cause considerable tissue damage (Todar, 2003).
3.9.2 Invasion
The bacteria invade and grow primarily due to their resistance to the host phagocytic response. The cell wall components directly activate multiple inflammatory cascades including the alternative pathway of complement activation, the coagulation cascade, and the cytokine cascade, inducing interleukin -1, -6 and tumor necrosis factor from macrophages and other cells (Todar, 2003).
In addition, as pneumococci begin to lyse in response to host defensins and antimicrobial agents, they release cell wall components, pneumolysin and other substances that lead to greater inflammation and cytotoxic effects. Pneumolysin and hydrogen peroxide kill cells and induce production of nitric oxide which may play a key role in septic shock (Todar, 2003).
During invasion, the interaction between the bacterial cell wall choline and the host PAF receptor G protein contribute to a state of altered vascular permeability. In the lung, this leads to arrival of n inflammatory exudates. At first, a serous exudate forms. This is followed by the arrival of leukocytes, thereby making the switch from a serous to purulent exudates (Todar, 2003).
Pneumococci occasionally are able to directly invade endothelial cells. In vitro, pneumococci will adhere to and traverse an endothelial barrier over approximately 4 hours. If bacteremia occurs, the risk of meningitis increases. Pneumococci can adhere specifically to cerebral capillaries using the same pairings of choline to PAF receptor and CbpA to carbohydrate receptor. Thus, the bacteria subvert the endocytosis/recycling pathway of the PAF receptor for cellular transmigration. Once in the cerebrospinal fluid, a variety of pneumococcal components, particularly cell wall components, incite the inflammatory response (Todar, 2003).
3.10 Cultivation
Streptococcus pneumoniae is fastidious bacterium, growing best in 5% carbon dioxide. In all cases, growth requires a source of catalase (e.g. blood) to neutralize the large amount of hydrogen peroxide produced by the bacteria. In complex media containing blood, at 37ºC, the bacterium has a doubling time of 20-30 minutes (Todar, 2003).
S pneumoniae is fermentative aerotolerant anaerobe. It is usually cultured in media that contain blood. On blood agar, colonies characteristically produce a zone of alpha (green) hemolysis, which differentiates S pneumoniae from the group A (beta hemolytic) streptococcus, but not from commensal alpha hemolytic (viridans) streptococci which are co-inhabitants of the upper respiratory tract. Special tests such as bile solubility, optochin sensitivity must be routinely employed to differentiate the pneumococcus from Streptococcus viridans (Todar, 2003).
S pneumoniae is a very fragile bacterium and contains within itself the enzymatic ability to disrupt and to disintegrate the cells. The enzyme is called an autolysin. The physiological role of this autolysin is to cause the culture to undergo a characteristic autolysis that kills the entire culture when grown to stationary phase. Virtually all clinical isolates of pneumococci harbor this autolysin and undergo lysis usually beginning between 18-24 hours after initiation of growth under optimal conditions. Autolysis is consistent with changes in colony morphology. Colonies initially appear with plateau type morphology, and then start to collapse in the centers when autolysis begins (Todar, 2003).
3.11 Identification
Streptococcus pneumoniae is Gram positive elongated diplococcus but may also occur singly and in short chains. Individual cells are between 0.5 and 1.25 micrometer in diameter (Todar, 2003). This pneumococcus is non motile, non sporing and capsulated (non capsulated following culture). In Gram stained smears from specimens, the capsule can often be detected as an unstained empty area around the diplococcus (Kaijalainen, 2006).
Optimal growth occurs in an increased carbon dioxide atmosphere. On blood agar, S pneumoniae forms translucent or mucoid colonies, 1-2 mm in diameter following overnight incubation at 35-37ºC. In young culture, the colonies are raised but as the culture ages, the colonies become flattened, with a depressed central part and raised edges giving them a ringed appearance (draughtsmen colony). The pneumococcus shows alpha hemolysis i.e., colonies are surrounded by an area of partial haemolysis with a greenish discoloration in the medium (Cheesbrough, 2005).
The minimum criteria for the identification and distinction of pneumococci from other streptococci are bile solubility, optochin sensitivity, gram positive staining and α-hemolytic activity. Pneumococci cause alpha hemolysis on agar containing sheep blood. Under anaerobic conditions they switch to beta hemolysis caused by an oxygen labile hemolysin. Typically, pneumococci form a 14 mm zone of inhibition around a 5 µg Optochin disc, and undergo lysis by bile salts. This lysis depends upon the presence of an autolysin enzyme, LytA (Kaijalainen, 2006).
3.12 Distribution of pneumococcal serotypes
Distribution of pneumococcal serotypes associated with disease can vary according to several parameters, including geographic area, period of analysis, and age group (Teixeira, 2002). Results of surveillance in more industrialized countries suggest that serotype distribution associated with infection in children is different from that observed in adults.
Among the 90 different polysaccharide serotypes, some are more virulent than others. A small number of them, approximately 10 serotypes, are common in pneumococcal infections. Serotypes 1, 3, 4, 6, 7, 9, 14, 18, 19, and 23 are the most frequent in children’s disease. According to a recent review, serotypes 1, 3, 5, 6, 14, 19 and 23 are comprehensive types in invasive pneumococcal infections on several continents (Kaijalainen, 2006).
Information on the regional distribution of pneumococcal serotypes is essential for the development and use of appropriate pneumococcal vaccines in developing countries. Serotypes 1, 5, 7, 19, and 23 are commonly encountered in India (Siberry et al., 2001).
Determining the serotypes of S pneumoniae from different clinical specimens is important as the vaccine production is based on the most common serotypes (Ozalp et al., 2004).
Effective surveillance of pneumococcal disease and its serotypes is needed to accurately map the magnitude of the problem and help evaluate the impact of available vaccines (www.infectioncontroltoday.com).
Fortunately, new vaccines to prevent deadly pneumococcal infections are now available and widely used in many countries in North America and Europe. With systematic surveillance in place and a coordinated effort to introduce pneumococcal vaccines we could save millions of children’s lives and make a significant move towards meeting a key U.N. Millennium Development Goal of reducing child mortality by two-thirds by 2015 (www.infectioncontroltoday.com).
3.13 Genetics
S pneumoniae has a natural transformation system as a mechanism for genetic exchange. This process is of medical significance because it clearly underlies the explosion of antibiotic resistance in the bacterium over the past 20 years. For example, penicillin resistance is due to altered penicillin binding proteins (PBPs) which exhibit a low affinity for beta lactam antibiotics. Comparison of the nucleotide sequences encoding the PBPs in S pneumoniae and S mitis demonstrates that horizontal gene transfer has occurred between these two bacteria. In the upper respiratory tract of the host, horizontal exchange of genetic information could take place between strains of pneumococci that co-habitat or compete for dominance as normal flora (Todar, 2003).
S pneumoniae can also develop antibiotic resistance by the timeless process of mutation and selection. The bacterium has a relatively fast growth rate and achieves large cell densities in an infectious setting. These conditions not only favor the occurrence of natural transformation, but also the emergence of spontaneous mutants resistant to the antibiotic (Todar, 2003).
3.14 Antimicrobial susceptibility
Antibiotics with activity against pneumococci include Penicillin, Erythromycin, Co-trimoxazole, and Cephotaxime. Isolates should also be tested for sensitivity to Tetracycline, Chloramphenicol (Cheesbrough, 2005).
Penicillin resistant strains are becoming an increasing problem in tropical Africa, South Africa, and else where. At the present time, clinical laboratories are advised to screen all clinical isolates of S pneumoniae for penicillin resistance. Such screening can be best performed by the use of a disc containing 1 µg of Oxacillin (WHO, 1994).
Antibiotic-resistant S. pneumoniae peaks at 0-2 years: resistance to penicillin, erythromycin.(Infectious Diseases)(Streptococcus pneumoniae): An article from: Pediatric News
3.15 Antimicrobial resistance
Also of concern, is the increased emergence of antibiotic resistance, especially in the past decade. Multiple antibiotic resistant strains of S pneumoniae that emerged in the early 1970s in Papua New Guinea and South Africa were thought to be a fluke, but multiple antibiotic resistance now covers the globe and has rapidly increased since 1995 (Todar, 2003).
Resistance to penicillin in S pneumoniae is increasing throughout the world. The problem is particularly common in Spain, Eastern Europe, South Africa, South America, New Guinea and Korea where resistance up to 30-50% is commonly reported (Catterall, 1999).
Increases in penicillin resistance have been followed by resistance to cephalosporins and multidrug resistance. The incidence of resistance to penicillin increased from <0.02 in 1987 to 3% in 1994 to 30% in some communities in the united states and 80% in regions of some other countries in 1998. Resistance to other antibiotics has emerged simultaneously: 26% resistant to Trimethoprim-sulphamethoxazole, 9% resistant to cefotaxime, 30% resistant to macrolides, and 25% resistant to multi drugs. Resistant organisms remain fully virulent but seem to have arisen in less than 10 serotypes. Serotypes 6A, 6B, 9V, 14, 19A and 23F are included in the vast majority of resistant strains (Todar, 2003). While antimicrobial resistance spawned by indiscriminate antibiotic usage in developing countries has received much attention, less understood are the epidemiology of antibiotic resistance and factors contributing to regional differences. Prevalence rates of resistance among nasopharyngeal or blood stream isolates of S pneumoniae and H influenzae from children in developing countries have been recently reviewed. The majority of S pneumoniae in South Asia are now Cotrimoxazole resistant- raising the question of whether W.H.O. ARI program should shift from Cotrimoxazole to more expensive Amoxicillin for treatment (Zaidi, 2003). Penicillin resistance among pneumococcal isolates in South Asia has also emerged and is gradually increasing, with 5-10% of isolates currently resistant. In Pakistan, Cotrimoxazole therapy has increasingly failed; two studies have found Cotrimoxazole to be ineffective in one-third of patients with pneumonia; and children under age of 1 year were especially susceptible to treatment failure (Zaidi, 2003). Comprehensive surveillance of drug resistance patterns in this microorganism is needed to guide prevention and control efforts. 3.16 Prevention of pneumococcal infection The increasing rate of antibiotic resistance in S pneumoniae complicates the elimination of pneumococci by therapy and strongly supports the application of new vaccine strategies. Preventive strategies for pneumococcal infection include use of the 23-valent polysaccharide pneumococcal vaccine for individuals older than 2 years of age, and routine immunization of infants and children with the 7-valent polysaccharide-protein conjugate pneumococcal vaccine. As vaccine coverage (i.e. immunization rate) in children and adults increase, the disease burden of invasive pneumococcal infections decreases (Weber and Rutala, 2003). 3.16.1 Anti-pneumococcal vaccine In 2002, 11 million infants living in developing countries died from vaccine preventable diseases. Pneumococcal infection accounts for more deaths than any other vaccine-preventable bacterial disease. Those most commonly at risk for pneumococcal infection are children between 6 months and 4 years of age and adults over 60 years of age (Dejsirilert et al., 1999). Capsular polysaccharide is the basis of the current anti pneumococcal vaccine. New vaccines to prevent deadly pneumococcal infections are now available and widely used in many countries in North America and Europe (www.infectioncontroltoday.com). Polysaccharide (PS) vaccine Given the 90 different capsular types of pneumococci, a comprehensive vaccine based on polysaccharide alone is not feasible. Thus, vaccines based on a subgroup of highly prevalent types have been formulated (Todar, 2003). The number of serotypes in the vaccine has increased from four in 1945, to 14 in the 1970s, and finally to the current 23-valent (polysaccharide vaccine) formulation (one dose 0.5 ml of the 23-valent vaccine contains 25 µg capsular polysaccharide antigen of each serotypes 1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F) (www.cdc.com, 1998). These serotypes represent 85%-90% of those that cause invasive disease in western industrialized countries and the vaccine efficacy is estimated at 60%. However, underutilization of the vaccine is so extensive that the pneumococcus remains the most common infectious agent leading to hospitalization in all age groups. This is further complicated by the fact that polysaccharides are not immunogenic in children under the age of 2 years where a significant amount of disease occurs (Todar, 2003). The currently available pneumococcal vaccines are manufactured by both Merck and Company, Inc. (Pneumovax 23) and Lederle Laboratories (Pnu-Imune 23). These vaccines were licensed in the United States in 1983 (www.cdc.com, 1998). Protein-polysaccharide (Conjugate) vaccine Over the past 15 years, several vaccine manufacturers have developed pneumococcal conjugate vaccines in which a number of S pneumoniae PS are covalently coupled to a protein carrier. Conjugate vaccines elicit higher antibody levels and a more efficient immune response in infants, young children and immunodeficient persons than the PS vaccines. Moreover, these vaccines suppress nasopharyngeal carriage of the pathogen and reduce bacterial transmission in the community through herd immunity, which adds considerable value to their implementation. Conjugate vaccines immunization followed by PS vaccine boosting might provide a foundation for lifelong protection against pneumococcal disease (WHO, 2006). Furthermore, the world wide increase in penicillin resistance among pneumococci and the limited use of the pneumococcal vaccine suggest that morbidity and mortality from pneumococcal disease may increase. CHAPTER IV 4 Materials and Methods 4.1 Materials Details of the materials used during the study are provided in Annex II, III, IV and V. 4.1.1 Equipments Biological Safety Cabinet Dalton Incubator Sakura: IF-3B, Tiyoda Manufacturing Company, Japan. Autoclave Sakura Neoclave ASV 2402, Tiyoda Hot Air Oven Sakura Clinioven TF-21 Microscope Olympus, PM-10 ADS; Olympus Optical Co. Ltd., Japan. Water Distillation Plant Adventec. GS-200, Japan. Weighing Machine Chyo JL- 200; Chyo Balance Co. Ltd., Japan. Refrigerator Toshiba Company, Japan Anaerobic Gas Jar Oxoid 4.2 Methodology 4.2.1 Study design The study was hospital based prospective study for which nasopharyngeal swab specimens were collected from the children attending OPD of KCH and all the processing were conducted in Health Research Laboratory, IOM, TUTH. All together 188 children attending KCH between February 2007 to September 2007 were involved. Consent was taken from guardian of every child and also from the child whenever appropriate or possible prior to enrollment. All the guardians were asked for the information as specified in the prepared questionnaire. 4.2.2 Sample collection After obtaining consent, nasopharyngeal swab specimen was collected using specifically designed pediatric sized swab of thin flexible aluminum shaft tipped with Dacron polyester. The flexible swab was inserted through one nasal aperture into the posterior wall of the nasopharynx. Once the swab was in place, it was slightly rotated and left for about 10 seconds to saturate the tip and removed slowly. Once a swab specimen was collected, it was placed in a tube of STGG medium. The excess wire handle was cut off from the swab using scissors sterilized with an alcohol wipe leaving the swab itself in the medium. The specimens were processed within 3 hours of collection following standard laboratory procedures. 4.2.3 Culture In the laboratory, the specimens were vortexed for 20-30 seconds to disperse organisms from the swab tip. Each specimen was inoculated on blood agar plate with 7% sheep blood. The inoculated plates were then incubated overnight at 37º C in CO2 enriched atmosphere. 4.2.4 Examination of culture plate Culture plates after overnight incubation were looked for the growth of S pneumoniae characterized by small, grayish mucoid colonies surrounded by greenish zone of hemolysis. One or two typical colony was picked and sub-cultured on blood agar plate with 7% sheep blood to obtain pure culture for further processing. 4.2.5 Identification of S pneumoniae The isolates were identified following Bergey’s Manual of Systematic Bacteriology. Colony of S pneumoniae appeared as small, grayish mucoid surrounded by greenish zone of haemolysis. Young colonies appeared raised while as the culture aged; colony became flattened at center with raised edges. On Gram staining, they appeared Gram positive elongated diplococcus and some occurred singly and in short chain under microscope. S pneumoniae was further confirmed by sub-culturing the isolate on BA and placing an Optochin disc (5 µg) on the primary streak line. 14 mm zone of inhibition around the disc after incubation at 37ºC for 24 hours confirmed S pneumoniae. It was also bile soluble. 4.2.6 Antibiotic susceptibility test Antibiotic susceptibility test of the isolated pneumococci was performed by modified Kirby-Bauer Disc Diffusion Technique. For the test, a suspension of pure overnight culture of the organism was prepared in Mueller-Hinton broth with its turbidity equivalent to 0.5 McFarland standard and it was swabbed on entire surface of Mueller-Hinton Agar plate with 7% sheep blood using sterile cotton swab. 6 antibiotic discs were placed around the edge of the plate and incubated overnight at 37ºC in CO2 enriched atmosphere. Diameter of zone of inhibition around each disc was measured and interpreted according to NCCLS recommendation. Zone size interpretative chart is given in Appendix VII. 4.2.7 Serotyping Serotyping of the isolated pneumococci was done by coagglutination method using Pneumotest (pneumococcal antisera) kit. The Pneumotest kit contains 12 pooled sera (A to F plus H, and P to T). Capsular polysaccharide of pneumococci is the basis of this serotyping technique. It is based on capsular reaction due to interaction between pneumococcal capsular polysaccharide and its homologous antibody. A positive reaction is indicated by the bacterial coagglutination as a result of an in situ immunoprecipitation. The procedures of serotyping and interpretation of result are given in Appendix VIII and IX. 4.2.8 MIC testing Minimum Inhibitory Concentration (MIC) of Oxacillin resistant isolates was determined against penicillin by E-test. The procedure of determining MIC by E-test is given in Appendix X. 4.2.9 Quality control It is of utmost importance to perform quality control throughout the study to obtain results that are both reliable and desirable. So, quality control was applied at different level during the study. • Samples were processed on the same day of collection as soon as possible. • All the processing of samples was performed aseptically within the biological safety cabinet. • Freshly prepared media were used every time and each batch of media was put into quality check. For that a freshly prepared media plate was incubated with ATCC 49619 and another was incubated uninoculated. Growth obtained on the inoculated plate helped to make out the type of growth to look for whereas absence of growth on uninoculated plate clearly indicated that the media was sterile. In this way, ATCC 49619 was used as control strain for both identification and antibiotic susceptibility test. CHAPTER V 5 Result Nasopharyngeal swab specimens from 188 children attending OPD of KCH were obtained randomly after obtaining informed consent from their parents. The collected samples were processed in the Health Research Laboratory, IOM, TU. The findings of the study were compiled and then analyzed with the chi-square statistic. 5.1 Pattern of result Of all 188 specimens investigated for S pneumoniae, 65 (34.57%) of them showed the growth of S pneumoniae and 123 (65.43%) were S pneumoniae negative as shown below in Figure 1. Figure 1: Subjects enrolled and gender wise distribution of S pneumoniae among them 5.2 Gender wise distribution of S pneumoniae Out of 65 positive cases, 33 (50.77%) were male and 32 (49.23%) were female. Gender wise distribution of nasopharyngeal carriage of S pneumoniae was statistically not significant (P > 0.05) (Table 1).
Table 1: Gender wise distribution of S pneumoniae
Gender Number Percentage
Male 33 50.77%
Female 32 49.23%
Total 65 100%
5.3 Prevalence of S pneumoniae colonization in different age groups
Nasopharyngeal carriage rate was highest among children of age group 2 to 12 months followed by age group 37 to 48 months, 13 to 24 months, 49 to 60 months and 25 to 36 months. The result showed that nasopharyngeal carriage of S pneumoniae was statistically significant (P < 0.05) among children of age group 2 to 12 months (Table 2). Table 2: Age wise distribution of S pneumoniae Age (in months) Number Percentage 2-12 34 52.31% 13-24 8 12.31% 25-36 5 7.69% 37-48 11 16.92% 49-60 7 10.77% Total 65 100% 5.4 Distribution of S pneumoniae in relation to type of cooking stove Nasopharyngeal carriage rate of S pneumoniae was highest among the children from the family using firewood cooking stove 47.69% followed by kerosene cooking stove 29.23% and gas cooking stove 23.08%. The association between type of cooking stove and carriage is statistically significant (P < 0.05) (Table 3). Table 3: Distribution of S pneumoniae in relation to type of cooking stove Type of cooking stove Number Percentage Firewood 31 47.69% Kerosene 19 29.23% Gas 15 23.08% Total 65 100% 5.5 Distribution of S pneumoniae according to family type Nasopharyngeal carriage rate of S pneumoniae was found to be highest among children from extended family (50.77%) in comparison to children from nuclear family (49.23%) however, it was not statistically significant (P > 0.05) (Table 4).
Table 4: Distribution of S pneumoniae according to family type
Family type Number Percentage
Extended 33 50.77%
Nuclear 32 49.23%
Total 65 100%
5.6 Distribution of S pneumoniae in relation to the presence of other children in the family
Nasopharyngeal carriage rate of S pneumoniae was same in children from family where there was no other child and where there was only one other child accounting for 33.85% each. Likewise, the carriage rate was 32.3% in children from family where number of other children was equal to or greater than 2. The association between number of other children and carriage rate was statistically significant (P < 0.05) (Table 5). Table 5: Distribution of S pneumoniae in relation to the presence of other children in the family Number of other child in the family Number Percentage 0 22 33.85% 1 22 33.85% ≥2 21 32.3% Total 65 100% 5.7 Distribution of different serotypes of the isolated S pneumoniae All S pneumoniae isolates were serotyped by coagglutination method using Pneumotest (pneumococcal antisera) kit. Among 65 S pneumoniae isolates, 5 were Non-Typeable (NT). Remaining 60 isolates were found to belong to 16 different serotypes. Frequency of occurrence of different serotypes is given in descending order: serotype 19 and serotype 6: 18% each, serotype 15: 9%, serotype 14: 8%, serotype 23 and serotype 11: 6% each, serotype 20, serotype 7 and serotype 3: 5% each, serotype 12: 3% and serotype 18, serotype 17, serotype 10, serotype 9, serotype 8 and serotype 5: 2% each (Table 6). Table 6: Distribution of different serotypes of the isolated S pneumoniae Serotypes Number Percentage 19 12 18% 6 12 18% 15 6 9% 14 5 8% 23 4 6% 11 4 6% 20 3 5% 7 3 5% 3 3 5% 12 2 3% 18 1 2% 17 1 2% 10 1 2% 9 1 2% 8 1 2% 5 1 2% Non-Typeable 5 8% Total 65 100% 5.8 Antimicrobial susceptibility pattern of S pneumoniae All 65 S pneumoniae isolates were tested against Cefotaxime, Cotrimoxazole, Chloramphenicol, Erythromycin, Tetracycline and Oxacillin using modified Kirby-Bauer disc diffusion method. Of them, 100% of these isolates were susceptible to Cephotaxime and Chloramphenicol, 98.46% were susceptible to Erythromycin. 78.46% were susceptible to Tetracycline and 12.31% were moderately susceptible to Tetracycline. On the other hand, 49.23% and 10.77% were susceptible and moderately susceptible respectively to Cotrimoxazole. 6.15% were found to be resistant to Oxacillin. Table 7: Antimicrobial susceptibility pattern of S pneumoniae Antibiotic Susceptible Moderately susceptible Resistant Total Number Percentage Number Percentage Number Percentage Number Percentage Cephotaxime 65 100% 0 0 0 0 65 100% Chloramphenicol 65 100% - - 0 0 65 100% Cotrimoxazole 32 49.23% 7 10.77% 26 40% 65 100% Erythromycin 64 98.46% 0 0 1 1.54% 65 100% Tetracycline 51 78.46% 8 12.31% 6 9.23% 65 100% Oxacillin 55 84.62% - - 10 15.38% 65 100% 5.9 MIC testing Oxacillin resistant isolates were tested against Penicillin using E-test strip. MIC of Penicillin of two Oxacillin resistant isolates was found moderately susceptible while that of remaining eight Oxacillin resistant isolates was found susceptible. CHAPTER VI 6 Discussion and conclusion 6.1 Discussion Many of the bacteria that cause ARI can be isolated as a part of the normal flora of healthy people. Streptococcus pneumoniae is a major community acquired pathogen responsible for vast majority of diseases particularly in young children below 5 years of age. Bacterial colonization of nasopharynx starts immediately after birth and continues throughout life with small changes and S pneumoniae also belongs to normal flora which is easily transmitted. Under certain circumstances, these colonizing microorganisms go on to cause disease. In this study, 188 nasopharyngeal swab specimens were collected from children attending OPD of KCH to determine colonization of nasopharynx by S pneumoniae. Of the total 188 nasopharyngeal swab specimens processed, only 65 (34.57%) of them showed the growth of S pneumoniae. This is supported by the study carried out by Todar in 2003, in which, nasopharyngeal colonization with pneumococci occurred in 40% of the cases. Among 65 subjects positive for S pneumoniae colonization, 33 (50.77%) were male and 32 (49.23%) were female. This showed that there was no correlation between gender and nasopharyngeal carriage (P > 0.05). This may be because, unlike in case of adults where males are more prone to ARI due to mobility and other predisposing factors such as smoking, children below 5 years of age stays at home irrespective of their gender. This can be correlated with findings of Malla et al., (2005) who reported similar results: 49% in female and 51% in male.
Nasopharyngeal carriage rate was found to be highest among children of age group 2-12 months accounting for 52.31%. The prevalence rate was found to be 16.92%, 12.31%, 10.77% and 7.69% in children of age group 37-48, 13-24, 49-60 and 25-36, respectively. The result of this study suggested a strong correlation between age of children and nasopharyngeal colonization with pneumococci (P < 0.05). High prevalence of pneumococci carriage in children belonging to 2-12 months age group is mainly due to close and attached mother to child relationship. Children of this age group are more in contact with their mother than children of other age group. As such, children of this age group mostly acquire their normal flora from their own mother according to the results of the study carried out by Catterall (1999). The carriage rate decreased with increase in age of child up to 36 months. It is attributable to decreased child to mother relationship with increase in age of child in comparison to 2 to 12 month’s age group. Children from 2 to 24 months of age represent preschool children and children attending child day care centers and nurseries among which carriage rates are highest. Children of age group 2 to 24 months contributed to highest carriage rate which was 64.62%. Carriage rate amongst children from 37 from 60 months of age is dependent on likelihood of their contact with other children according to Catterall (1999). Nasopharyngeal carriage of S pneumoniae was highest, i.e. 47.69% among the children from family using firewood cooking stove followed by kerosene cooking stove 29.23% and gas cooking stove 23.08%. This clearly reflected strong correlation between carriage rate and type of cooking stove which is also statistically significant (P < 0.05). Cooking with firewood stove results in highest level of indoor air pollution followed by kerosene stove and gas stove. Smoke in the home from cooking stove is also an important contributing factor of nasopharyngeal colonization. Women and children are predominantly the victims of smoke produced in kitchen while cooking. Women spend at least three hours a day in kitchen and children under five years of age spend most of the time with their mothers. So they are also exposed to smoke (Malla et al., 2005). Indoor pollution has considerable impact on pneumococcal carriage and its rates according to Cant et al, (2002) and Kaijalainen (2006). The respiratory tract is directly exposed to many potential pathogens via smoke, soot and dust that are inhaled from the air as stated by WHO (2003). Family type is also considered in this study because its type represents the number of family members to whom the child in the family is exposed. Extended family has large number of family members whereas nuclear family has few family members. In this study, carriage rate of S pneumoniae was found to be 50.77% in children from extended family and 49.23% from nuclear family. Here, although carriage rate was highest in children from extended family, there was no significant difference in carriage rate between children from extended and nuclear family. The most probable reason behind it may be because the children under 5 years of age spend most of their time with their mothers rather than with other members of the family. From this, we can conclude that there is no correlation between nasopharyngeal carriage and family type (P > 0.05).
Although carriage of S pneumoniae was very similar among families where there were no other children, where there were only one child and where there were two and more than two children, there is association between the carriage and number of other children in the family. These are also associated statistically (P < 0.05).
In the family where there was no other child, obviously the child gets greater chance to be with its mother. In the family where there is another one child, due to less age difference between two, both of them have nearly equal chance to spend with their mother. So carriage was similar. In the family where there are two and more other children, the carriage rate was higher. It may be due to transmission of pneumococci among children.
The nasopharyngeal isolates of S pneumoniae were serotyped by coagglutination method using Pneumotest (pneumococcal antisera) kit. The isolates were found to belong to 16 different serotypes. Frequency of occurrence of different serotypes is given in descending order: serotype 19 and serotype 6: 18% each, serotype 15: 9%, serotype 14 8%, serotype 23 and serotype 11: 6% each, serotype 20, serotype 7, and serotype 3: 5% each, serotype 12: 3% and serotype 18, serotype 17, serotype 10, serotype 9, serotype 8 and serotype 5: 2% each. Remaining 8% were found to be Non-Typeable.
Information on the regional distribution of pneumococcal serotype is essential for the development and use of appropriate pneumococcal vaccine in developing countries. Determining the serotype of S pneumoniae from different clinical specimens is important as the vaccine production is based on the most common serotypes (Siberry et al., 2001 and Ozalp et al., 2004).
Serotype 1 is regarded as most invasive. However, this serotype was not encountered in this study. Serotypes 1, 3, 5, 6, 14, 19, and 23 are considered comprehensive types in invasive pneumococcal infections (Kaiijalainen, 2006). Except serotype 1, all serotypes considered comprehensive type in invasive pneumococcal infections were found in the study.
Serotypes 19 and 6 were found to be the most common serotypes in present study. Other common serotypes included 15, 14, 23, 11, 20, 7, 3 and 12.
Serotypes 19, 6, 15, 23, 9, 11, 8, 7, 17, 20, 22 were commonly involved in nasopharyngeal colonization in children according to the report by Ozalp et al., (2004). Serotypes 3, 19, 23, 6, 14 were most common nasopharyngeal isolates in children in the study carried out by Marchisio et al., in 2002.
All the isolated pneumococci were found to be susceptible to Cephotaxime and Chloramphenicol. Erythromycin is also effective drug of choice since only 1.54% was found to be resistant to it. Tetracycline can also be used for therapy since only 9.23% isolates were found to be resistant to it.
Cotrimoxazole was recommended by W.H.O. to treat against infection caused by S pneumoniae due to its lesser side effects, lower cost and easy availability. However, in the current study, Cotrimoxazole showed lowest susceptibility with 40% resistance. Also in a similar study, previously carried out by Malla et al., in 2005, it was the least effective drug against S pneumoniae. In Pakistan, two studies have found Cotrimoxazole to be ineffective in one third of patients with pneumonia; and children under age of 1 year were especially susceptible to treatment failure. The majority of S pneumoniae in South Asia are now Cotrimoxazole resistant- raising the question of whether W.H.O. should shift from Cotrimioxazole to more expensive Amoxicillin for treatment (Zaidi, 2003).
The key factor behind the emergence of Cotrimoxazole resistant pneumococci is unnecessary use of antibiotic for viral respiratory infections because of widespread confusion over the difference between viral and bacterial respiratory infections. Also, antibiotics are freely available in the market without prescriptions. Cotrimoxazole is the cheapest antibiotic which is widely available in the market in comparison to other antibiotics and it is most commonly used by people without discrimination between viral and bacterial respiratory infections.
In the current study 15.38% of the isolates were found to be Oxacillin resistant. In study carried out by Malla et al., in 2005, 5.12% of nasopharyngeal isolates of pneumococci were found to be Oxacillin resistant. There has been a slight increase, in this study, in Oxacillin resistance. Clinical laboratories are advised to screen all important isolates of S pneumoniae for Penicillin resistance. Therefore, though Oxacillin is not used in therapy, it is used in vitro antimicrobial susceptibility testing of S pneumoniae for predicting resistance of S pneumoniae to Penicillin because of its greater resistance to deterioration during its storage and it provides the most reproducible results.
On performing E-test of Oxacillin resistant strains against Penicillin, MIC of two of the Oxacillin resistant pneumococcal isolates was found moderately susceptible and remaining was found to be susceptible to Penicillin.
Though Oxacillin resistance rate is not large, the value is alarming. Though disc sensitivity testing accurately reflects whether an organism is resistant to most antimicrobial agents, disc testing of Oxacillin resistance for S pneumoniae is not sufficient to distinguish between complete and partial resistance since it does not distinguish penicillin intermediate resistant strains from strains that are penicillin resistant. MIC testing of isolates identified by disc as resistant is needed to quantify the level of resistance of S pneumoniae to Penicillin. The higher the MIC, the more likely treatment is to be ineffective (WHO). Therefore MIC of Oxacillin resistant isolates was determined by E-test against Penicillin.
Penicillin resistance among pneumococcal isolates in South Asia has also emerged and is gradually increasing, with 5-10% of isolates currently resistant. In Korea, it is 25-30% (Zaidi, 2003).
6.2 Conclusion
Nasopharyngeal carriage study of Streptococcus pneumoniae was carried out among children attending Kanti Children’s Hospital and the carriage rate was found to be approximately 35%.
CHAPTER VII
7 Summary and Recommendation
7.1 Summary
1. Nasopharyngeal colonization with S pneumoniae was found in approximately 35% of the studied population and colonization rate was similar in both male and female.
2. The colonization rate was greater in the children from the family using firewood as cooking stove.
3. Children of age group 2 to 24 months contributed to highest carriage rate.
4. Among the isolated pneumococci, 5 were found to be Non-Typeable and remaining were found to belong to 16 different serotypes.
5. On performing antibiotic susceptibility test, Cephotaxime, Chloramphenicol, Erythromycins were found to be most effective antibiotics against the isolates of pneumococcus whereas Cotrimoxazole showed least susceptibility.
6. On performing MIC testing of the Oxacillin resistant isolates by E-test, two of the isolates were found to be moderately susceptible to Penicillin and remaining eight isolates were found susceptible. From this, we could conclude that Penicillin resistance had not evolved in the isolates of S pneumoniae in children.
7.2 Recommendations
Following recommendations are made based on this study for further study:
1. Current study was hospital based study. This study can also be carried out in community and also in children attending nurseries and kindergarten as a comparative study among themselves and between two groups.
2. The study on seasonal variation and impact of season on nasopharyngeal colonization can also be carried out.
3. It is also possible to determine MIC of Cotrimoxazole resistant S pneumoniae as recommended by WHO to quantify the level of resistance as disc testing of Cotrimoxazole resistance for S pneumoniae isolates is not sufficient to distinguish between complete and partial resistance.
4. It is also possible to compare nasopharyngeal colonization of mother and child with pneumococci and accessing epidemiology in the two.
CHAPTER VIII
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Pneumococcal vaccination may limit resistance. (Antibiotics and S.Pneumoniae).(Brief Article): An article from: Family Practice News
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1 INTRODUCTION
Respiratory system can be divided into upper and lower tracts. The respiratory and gastrointestinal tracts are the two major connections between the interior of the body and the outside environment. The respiratory tract is the pathway through which the body acquires fresh oxygen and removes unneeded carbon dioxide. It begins with the nasal and oral passages (Forbes et al., 2002).
The upper respiratory tract is frequently the site of general and localized infections. It is the primary site of infection for most viral diseases, which are spread by sneezing, coughing or direct contact with materials contaminated by respiratory secretions. Although the majority of such symptoms are viral in origin, secondary bacterial infection may often follow, particularly in the very young and malnourished. Resident bacteria in the upper respiratory tract such as Haemophilus influenzae, Streptococcus pneumoniae and Streptococcus pyogenes are the most common causes (Greenwood et al., 2003).
Respiratory tract infection is the major health problem in developing countries. Infection of the respiratory tract is the most frequent and important cause of short term illness in the population. It is frequently the first infection to occur after birth, and too often the final illness before death (especially pneumonia) (Dawadi et al., 2005).
The respiratory tract is the most common site for infection by pathogens. This site becomes infected frequently because it comes into direct contact with the physical environment and is exposed to the microorganisms in the air (Chantler and Griffith, 2004).
Acute respiratory tract infections (ARIs) are one of the most important causes of morbidity and mortality in children throughout the world. More than 4 million children under 5 years of age are estimated to die from ARI every year. This represents about 30 % of 14.25 million deaths of children under 5 years of age that occur in the developing world each year (Teixeira, 2002).
ARIs are estimated to be responsible for one third of all childhood deaths in developing countries. It is estimated that Bangladesh, India, Indonesia and Nepal together account for 40% of global acute respiratory infection mortality. These respiratory infections can manifest in any area of the respiratory tract, including the nose, middle ear, throat, voice box, air passage and lungs. As an infection of lungs, pneumonia is one of the major causes of ARI. About 90% of ARI deaths are due to pneumonia, which usually is bacterial in origin (WHO, 2000).
Approximately 2.6 million children under 5 years of age die annually of pneumonia predominantly in the developing world; approximately one half of these deaths are attributable to S pneumoniae either solely or in conjunction with a viral respiratory infection, malnutrition or HIV infection (O’brien et al., 2003).
Streptococcus pneumoniae are involved chiefly in the infections of upper and lower respiratory tracts. The pneumococci, in low numbers, is a part of the normal nasopharyngeal and oropharyngeal flora of many healthy persons and also children, which generally remains harmless unless it is provoked by a viral infections such as influenzae or the common cold to spread to the lower respiratory tract, middle ear, paranasal sinus or the blood. In this situation, pneumococci are secondary pathogen but may be primary pathogen in immunocompromised people.
Bacterial colonization of nasopharynx starts immediately after birth and continues throughout life with small changes. However, a major part of infections is caused by microbes like S pneumoniae, H influenzae, M catarrhalis, N meningitidis and Staphylococcus aureus, which originally belong to normal flora (Kaijalainen, 2006). Colonization frequently occurs without the development of disease (Catterall, 1999).
People usually carry pneumococci without symptoms, but carriage can also contribute to respiratory or even systemic disease. Several factors have considerable impact on pneumococcal carriage and its rates (Kaijalainen, 2006).
The bacteria S pneumoniae carried in the nasopharynx of children reflect the infection causing strains currently circulating in the community. So studies of the prevalence of different pathogens and their resistance patterns can provide useful indications for more rational therapeutic and preventive strategies. The symptomatic nasopharyngeal carriage of S pneumoniae is widely prevalent in young children and has been related to the development of disease and the spread of the pathogen. Furthermore, nasopharyngeal colonization by antibiotic resistant S pneumoniae has steadily increased over the last few years. Antibiotic resistant strains are more often carried by infants and young children than adults and belong to a limited number of serotypes that are also some of the most common cause of invasive pediatric diseases (Marchisio et al., 2002).
There was a belief among some commentators a generation ago that infectious disease was a problem that was well on the way to permanent resolution owing to the development of effective vaccines and antibiotics. However, such complacency has now completely disappeared (Denyer et al., 2005) and infectious diseases had still remained the leading cause of death all over the world.
Considering the above mentioned facts, this dissertation work as the partial fulfillment for Master Degree was conducted among the children attending Kanti Children’s Hospital in order to determine the prevalence of nasopharyngeal carriage of S pneumoniae, its antibiotic susceptibility pattern and distribution of its serotypes.
CHAPTER II
2 Objectives
This study was conducted with the following objectives
2.1 General objective
To assess sero-epiemiology of Streptococcus pneumoniae and determine its antibiotic susceptibility pattern in children less than 5 years of age attending out patient department of Kanti Children’s Hospital.
2.2 Specific objectives
1. To determine the prevalence of nasopharyngeal carriage of Streptococcus pneumoniae among children attending Kanti Children’s Hospital.
2. To isolate and perform antibiotic susceptibility test of Streptococcus pneumoniae from nasopharyngeal swab.
3. To serotype the isolates employing co-agglutination method.
4. To perform MIC for Oxacillin resistant strains.
CHAPTER III
3 Literature review
3.1 Respiratory tract infection
Respiratory tract infection is a major health problem in developing countries. Infection of the respiratory tract is the most frequent and important cause of short term illness in the population. It is frequently the first infection to occur after birth, and too often the final illness before death (especially pneumonia) (Dawadi et al., 2005).
Respiratory tract infections occur more frequently than they are reported and are often thought of as inconveniences of life that will pass away quickly (Dawadi et al., 2005). Respiratory tract infection, comprising a broad spectrum of diseases from self limiting acute bronchitis to severe pneumonia, is caused by a wide range of microbial pathogens (Hosker, 1994).
The human respiratory tract is exposed to many potential pathogens via the smoke, soot, and dust that are inhaled from the air. It has been calculated that the average individual ingests about 8 microorganisms per minute or 10,000 per day (WHO, 2003).
Children are smaller; their surface area is greater in comparison to the body weight therefore they tend to have higher metabolic rate and due to these reasons they have higher respiratory rate than the adults. So, this means they ingest more microorganisms than adult (www.medscape.com).
The respiratory tract is the most common site for infection by pathogens. This site becomes infected frequently because it comes into direct contact with the physical environment and is exposed to the microorganisms in the air (Chantler and Griffith, 2004).
3.2 Organisms present in the nasopharynx and oropharynx of healthy human
Possible pathogens Rarely pathogens
Acinetobacter spp
Viridans streptococci
Beta hemolytic streptococci
Streptococcus pneumoniae
Staphylococcus aureus
Neisseria meningitidis
Mycoplasma spp
Haemophilus influenzae
Haemophilus parainfluenzae
Moraxella (Branhamella) catarrhalis
Candida albicans
Herpes simplex virus
Enterobacteriaceae
Mycobacterium spp
Pseudomonas spp
Klebsiella ozaenae
Bacteroides spp
Peptostreptococcus spp
Actinomyces spp
Haemophilus aphrophilus
Entamoeba gingivalis Nonhemolytic streptococci
Staphylococci
Micrococci
Corynebacterium spp
Coagulase negative staphylococci
Lactobacillus spp
Veillonella spp
Spirochetes
Campylobacter spp
Source: Forbes et al, 2002
3.3 Epidemiology of ARI among children
Acute respiratory tract infections (ARIs) are one of the most important causes of morbidity and mortality in children throughout the world. More than 4 million children under 5 years of age are estimated to die from ARI every year. This represents about 30 % of 14.25 million deaths of children under 5 years of age that occur in the developing world each year (Teixeira, 2002).
According to the WHO Report 2000, the top five respiratory diseases account for 17.4% of all deaths and 13.3% of all Disability Adjusted Life Years (DALYs). Also, out of total acute respiratory disease, 20-24% of deaths are accounted for by lower respiratory tract infection (Verma, 1981).
Lower respiratory tract infections are the major burden of premature death and disability worldwide and as might be predicted, the burden is significantly greater in the developing countries compared with the developed world (Cant et al., 2002).
3.4 Respiratory tract infections in developing world
ARIs are estimated to be responsible for one third of all childhood deaths in developing countries. Although the incidence of ARI, at 5-9 episodes/child/year in the first 5 years of life, is the same in developed and developing countries, the incidence of acute lower respiratory tract infection (ALRI) is over 12- fold greater in developing countries (Cant et al., 2002).
Risk factors for progression from ARI to ALRI include young age (0-11 months), malnutrition (both macro- and micro- nutrients), lack of breast feeding, HIV infection and environmental factors such as crowding and indoor air pollution (Cant et al., 2002).
It is estimated that Bangladesh, India, Indonesia and Nepal together account for 40% of global acute respiratory infection mortality. These respiratory infections can manifest in any area of the respiratory tract, including the nose, middle ear, throat, voice box, air passage and lungs. As an infection of lungs, pneumonia is one of the major causes of ARI (WHO, 2000).
3.5 Causative agents of RTIs
It is clear that bacteria figure prominently in both primary and secondary roles in acute upper and lower respiratory tract infections. Many of the bacteria that cause ARI can be isolated as a part of the normal flora of healthy people. Under certain circumstances, these colonizing microorganisms go on to cause disease (Teixeira, 2002).
Bacterial infections of the respiratory tract can be grouped according to their symptomatology and anatomic involvement. Some of the causative agents are associated with specific syndromes (Teixeira, 2002).
Acute upper respiratory infections are usually benign, transitory and self limited, although some exceptions such as epiglottitis and laryngotracheitis may be severe diseases in small children and neonates. Most of the severe bacterial epiglottitis cases are caused by Haemophilus influenzae. Other severe bacterial infections of the upper respiratory tract are whooping cough (pertussis) caused by Bordetella pertusis and diphtheria caused by Corynebacterium diphtheriae. Pharyngitis, one of the most common bacterial infections, especially in the pediatric age group, is most often caused by Streptococcus pyogenes. H influenzae and S pneumoniae account for the vast majority of sinusitis cases. The most common bacterial pathogens recovered from middle ear of children with acute otitis media are S pneumoniae, H influenzae and Branhamella catarrhalis (Teixeira, 2002).
Pneumonia is the main lower respiratory tract infection, with characteristics much more severe than most of the upper ARI. About 90% of ARI deaths are due to pneumonia, which usually is bacterial in origin (WHO, 2000).
3.6 Diseases caused by Streptococcus pneumoniae
Serious pneumococcal infections occur throughout life, but young children under 5 years old (especially among those under 2 years old) and the elderly are at the highest risk for severe pneumococcal disease. Diseases caused by S pneumoniae include pneumonia (usually lobar type), paranasal sinusitis, otitis media, meningitis, bacteremia, conjunctivitis, etc. Furthermore, more than 90% of pneumococcal pneumonia deaths in children occur in developing countries and pneumococcal meningitis kills or disables over 40% of the children who get the disease (www.infectioncontroltoday.com).
Approximately 2.6 million children under 5 years of age die annually of pneumonia predominantly in the developing world; approximately one half of these deaths are attributable to S pneumoniae either solely or in conjunction with a viral respiratory infection, malnutrition or HIV infection (O’brien et al., 2003).
S pneumoniae continues to be a leading cause of pneumonia, meningitis and otitis media in persons of all ages. Pneumococci are also the most frequent cause of otitis media and bacteraemia and an important agent of sinusitis in children. There is evidence suggesting that most children experience some kind of pneumococcal infection. Approximately 80% of all children experience at least one attack of otitis media by the age of 3 years and pneumococci account for about half of these cases (Teixeira, 2002).
Streptococcus pneumoniae was identified as a major respiratory pathogen shortly after its isolation in 1881. Despite a century of intensive study and antibiotics which readily kill the organism, respiratory tract infections caused by the S pneumoniae remain a formidable problem. S pneumoniae is the commonest cause of community acquired pneumonia (Catterall, 1999).
3.6.1 Mode of transmission
Pneumococci are a part of the normal microbial flora of the nose and pharynx, particularly among young children and are easily transmitted. Infection usually occurs in a person already colonized with S pneumoniae, when the bacteria invade into the patient’s body. Person to person spread is rare, however S pneumoniae can be spread from person to person by inhalation of respiratory droplets (e.g., coughing, sneezing) from an infected person, by direct contact and indirectly by contact with articles such as clothing or tissues freshly soiled with respiratory secretions (Streptococcus pneumoniae, 2004).
Also, the transmission of pneumococci is increased during the course of other respiratory infections when secretions, coughing and sneezing are increased. Although transient nasopharyngeal colonization rather than disease is the normal outcome of exposure to pneumococci, bacterial infection following penetration of the mucosal layer, may occur in persons susceptible to the involved serotype (WHO, 2006).
3.6.2 Incubation period
Due to the fact that many infections arise from bacteria the patient is already carrying it is hard to identify an incubation period, however if a person develops an infection from a new exposure it is commonly within 1-3 days. It is also dependent upon how the illness manifests (Streptococcus pneumoniae, 2004).
3.6.3 Reservoir
S pneumoniae is commonly found in the upper respiratory tract of healthy people as a part of normal flora (Streptococcus pneumoniae, 2004).
3.7 Pneumococcus as a part of normal nasopharyngeal flora
Bacterial colonization of nasopharynx starts immediately after birth and continues throughout life with small changes. Normal nasopharyngeal bacterial flora develops during the first year of life and the number of bacterial species varies much. Normal flora has an important role in the prevention of infectious diseases. However, a major part of infections is caused by microbes like S pneumoniae, H influenzae, M catarrhalis, N meningitidis and Staphylococcus aureus, which originally belong to normal flora (Kaijalainen, 2006).
Pneumococcus has genetic properties allowing it to co-exist with other bacteria and inhibit competing intruders, and by producing hydrogen peroxide, it inhibits the growth of other bacteria such as H influenzae, M catarrhalis and N meningitidis (Kaijalainen, 2006).
3.7.1 Pneumococcal carriage rate
Nasopharyngeal colonization frequently occurs without the development of disease. Colonization can occur within hours of birth and by the 12th postnatal day, the carrier rate is similar to that of the babies’ mothers. Carriage rates are highest in preschool children, children attending child day care centers and nurseries while rates amongst adults depend on the likelihood of contact with other children (Catterall, 1999).
Recent studies during two outbreaks of pneumococcal pneumonia have shown that asymptomatic nasopharyngeal colonization with S pneumoniae frequently results in the production of circulating type specific antibody at levels which confer protection from pneumonia against that serotype. It therefore appears that, although aspiration of colonizing organisms during the first few weeks of colonization may lead to pneumonia, after that time most healthy adults are likely to be protected (Catterall, 1999). Invasive disease is most likely to occur soon after nasopharyngeal colonization with a newly acquired serotype rather than after long duration of carriage of that serotype (O’brien et al., 2003).
The rate of colonization appears to be seasonal, with an increased prevalence seen during the winter (Weber and Rutala, 2003).
Pneumococcus carriage develops among children more rapidly in developing countries compared with industrialized countries (O’brien et al., 2003).
3.7.2 Impacts on pneumococcal carriage rate
People usually carry pneumococci without symptoms, but carriage can also contribute to respiratory or even systemic disease. Several factors, such as age, geographical sites, socio-economic status, family size, indoor air pollution, number of siblings, day care, the presence of upper respiratory tract infection and overcrowded living conditions, have considerable impact on pneumococcal carriage and its rates (Kaijalainen, 2006).
A comparison of pneumococcal carriage rates between studies is difficult due to the variable methodological factors, such as the number and frequency of specimen collection, the quality of specimens and culture techniques. Generally, pneumococcal carriage is highest at the age of 2 years and it decreases over the years (Kaijalainen, 2006).
3.8 Bacterial determinants of virulence
3.8.1 Capsule
A capsule composed of polysaccharide completely envelops the pneumococcal cells. During invasion the capsule is an essential determinant of virulence. The bacterial capsule interferes with phagocytosis by preventing C3b opsonization of the bacterial cells i.e., by interference with binding of complement C3b to the cell surface (Todar, 2003).
The polysaccharide is non toxic and non inflammatory, and the capsule does not appear to engage any host defenses except for the induction of antibody mediated immunity. The pneumococcal capsule is not an antigenic disguise, and it does not impede the activities of underlying components, such as the cell wall and surface proteins, to engage the host defense systems. However, C reactive protein or antibodies to teichoic acid, both of which bind to the cell wall under the capsule, fail to opsonize encapsulated pneumococci (Todar, 2003).
90 different capsular types of pneumococci have been identified and form the basis of antigenic serotyping of the organism. Anti pneumococcal vaccines are based on formulations of various capsular (polysaccharide) antigens derived form the highly prevalent strains (Todar, 2003).
3.8.2 Cell wall (components)
The cell wall of S pneumoniae is roughly six layers thick and is composed of peptidoglycan with teichoic acid attached to approximately every third N-acetylmuramic acid. Lipoteichoic acid is chemically identical to the teichoic acid but is attached to the cell membrane by a lipid moiety. Both the teichoic acid and the lipoteichoic acid contain phosphorylcholine; two choline residues may be covalently added to each carbohydrate repeat. This is an essential element in the biology of S pneumoniae since the choline specifically adheres to choline binding receptors that are located on virtually all human cells (Todar, 2003).
The pneumococcal cell wall is a collection of potent inflammatory stimuli. Challenge with cell wall components alone can recreate many of the symptoms of pneumonia, otitis media and meningitis in experimental models. The phosphorylcholine decorating the teichoic acid and the lipoteichoic acid is a key molecule enabling invasion, and acts both as an adhesion and as a docking site for the choline binding proteins (CBPs). Other respiratory pathogens such as Haemophilus, Pseudomonas, Neisseria and Mycoplasma also have phosphorylcholine on lipopolysaccharide, proteins or fimbriae, suggesting a shared mechanism for invasion of the respiratory tract. Two host derived elements that recognize choline are platelet activating factor (PAF) receptor and the C reactive protein (Todar, 2003).
The peptidoglycan/teichoic acid complex of the pneumococcus is highly inflammatory. Smaller components of peptidoglycan progressively lose specific inflammatory activity. The cell wall directly activates the alternative pathway of the complement cascade, generating chemotaxins for leukocytes, and the coagulation cascade, which promotes a procoagulant state favoring thrombosis. In addition, peptidoglycan binds to CD14, a cell surface receptor known to initiate the inflammatory response for endotoxin. This induces a cytokine cascade resulting in production of interleukin- 1, -6 and tumor necrosis factor from human cells (Todar, 2003).
3.8.3 Surface proteins
On the basis of functional genomic analysis, it is estimated that the pneumococcus contains more than 500 surface proteins. Some are membrane associated lipoproteins, and others are physically associated with the cell wall. The latter includes five penicillin binding proteins (PBPs), two neuramanidases, and an IgA protease. A unique group of proteins on the pneumococcal surface is the family of choline binding proteins (CBPs) (Todar, 2003, Cattrerall, 1999).
Choline binding proteins
Twelve CBPs are noncovalently bound to the choline moiety of the cell wall and are used to “snap” various different functional elements onto the bacterial surface. The CBPs all share a common C terminal choline binding domain while the N-termini of the CBPs are distinct, indicating their functions are different. The CBP family includes such important determinants as PspA (protective antigen), Lyt A, B and C (three autolysins), and CbpA (adhesin) (Todar, 2003).
PspA (pneumococcal surface protein A)
PspA is a protective antigen with 10 choline binding repeats. PspA appears to inhibit complement mediated opsonization of pneumococci, and mutants lacking PspA have reduced virulence.
Autolysin LytA
It is responsible for pneumococcal lysis in stationary phase as well as in the presence of antibiotics. The protein has two functional domains: a C terminal domain with 6 choline binding repeats that anchor the protein on the cell wall, and a N terminal domain that provides amidase activity.
Autolysin LytB is a glucosaminidase involved in cell separation.
Autolysin LytC exhibits lysozyme like activity.
CbpA
It is a major pneumococcal adhesin. It has 8 choline binding repeats. The adhesin interacts with carbohydrates on the pulmonary epithelial surface carbohydrates. CbpA deficient mutants are defective in colonization of the nasopharynx and fail to bind to various human cells in vitro. CbpA also has been reported to bind secretory IgA and complement component C3.
Schematic figure of the known virulence factors of Streptococcus pneumoniae including their main functions and cellular location. Only factors with proven virulence are included
Source: Catterall, 1999
3.8.4 Hemolysins
In addition to surface associated virulence determinants, pneumococci secrete exotoxins. Two hemolysins have been described, the most potent of which is pneumolysin.
Pneumolysin is stored intracellularly and is released upon lysis of pneumococci by autolysin. Pneumolysin binds to cholesterol and thus can indiscriminately bind to all cells without restriction to a receptor. This protein assembles into oligomers to form transmembrane pores which ultimately lead to cell lysis. Pneumolysin can also stimulate the production of inflammatory cytokines, inhibit beating of the epithelial cell cilia, inhibit lymphocyte proliferation, decrease the bacterial activity of neutrophils, and activate complement.
A second hemolysin activity has been described but has not been identified. In addition, pneumococci also produce hydrogen peroxide in amounts greater than human leukocytes produce. This small molecule is also a potent hemolysin.
3.9 Pathogenesis (Transition from colonization to pneumonia and other invasive diseases)
Although S pneumoniae exists in encapsulated and unencapsulated forms, only encapsulated strains have been isolated from clinical material. The importance of the capsule in pneumococcal virulence has been established. However, the capsule itself is not toxic. Composed of one of 90 serologically distinct polysaccharides, the virulence of the capsule lies mainly in its antiphagocytic properties. The level of virulence is determined more by the chemical nature of the capsule then by its size (Catterall, 1999).
The factors which permit pneumococci to spread beyond the nasopharynx are poorly characterized and are likely to vary depending on the virulence of the organism, the state of the host’s defences, and the existence of preceding viral infection. Spread to the lungs probably occurs by aspiration which is aided by impairment of the cough reflex, by increased production of mucus (in which pneumococci also replicate), and by impairment of the mucocilliary escalator. Whilst all of these can be caused by host related disorders, the pneumococcus itself can contribute by pneumolysin dependent disruption of the epithelial type junctions which are essential for the production of mucus. Both influenza virus and adenovirus enhance in vitro adherence of S pneumoniae to respiratory tract epithelial cells (Catterall, 1999).
3.9.1 Colonization
Pneumococci adhere tightly to the nasopharyngeal epithelium through a variety of mechanisms involving the specific interaction between bacterial surface adhesions and epithelial cell receptors. Infection results when colonizing bacteria invade tissue and escape phagocytic defense mechanisms. This commonly occurs when bacteria are transported into the eustachian tube, sinuses, or bronchi (Weber and Rutala, 2003). Passage of pneumococci up the eustachian tube is accompanied by bacterial induced changes in the surface receptors of the epithelial cell, particularly by neuraminidase. Inflammation in the middle ear is caused by pneumococcal cell wall components, and pneumolysin inflicts major cytotoxicity on ciliated cells of the cochlea (Todar, 2003).
Upon reaching the lower respiratory tract by aerosol, pneumococci bypass the ciliated upper respiratory epithelial cells unless there is damage to the epithelium. Instead, they progress to the alveolus and associate with specific alveolar cells which produce a choline containing surfactant (Todar, 2003).
Experimentally, in healthy tissues, it requires approximately 100,000 bacteria/ml to trigger an inflammatory response. However, if a pro inflammatory signal is supplied, inflammation ensues with as few as 10 bacteria. This signal is a cytokine in experimental systems or an intercurrent viral infection in clinical situations. The inflammatory response can cause considerable tissue damage (Todar, 2003).
3.9.2 Invasion
The bacteria invade and grow primarily due to their resistance to the host phagocytic response. The cell wall components directly activate multiple inflammatory cascades including the alternative pathway of complement activation, the coagulation cascade, and the cytokine cascade, inducing interleukin -1, -6 and tumor necrosis factor from macrophages and other cells (Todar, 2003).
In addition, as pneumococci begin to lyse in response to host defensins and antimicrobial agents, they release cell wall components, pneumolysin and other substances that lead to greater inflammation and cytotoxic effects. Pneumolysin and hydrogen peroxide kill cells and induce production of nitric oxide which may play a key role in septic shock (Todar, 2003).
During invasion, the interaction between the bacterial cell wall choline and the host PAF receptor G protein contribute to a state of altered vascular permeability. In the lung, this leads to arrival of n inflammatory exudates. At first, a serous exudate forms. This is followed by the arrival of leukocytes, thereby making the switch from a serous to purulent exudates (Todar, 2003).
Pneumococci occasionally are able to directly invade endothelial cells. In vitro, pneumococci will adhere to and traverse an endothelial barrier over approximately 4 hours. If bacteremia occurs, the risk of meningitis increases. Pneumococci can adhere specifically to cerebral capillaries using the same pairings of choline to PAF receptor and CbpA to carbohydrate receptor. Thus, the bacteria subvert the endocytosis/recycling pathway of the PAF receptor for cellular transmigration. Once in the cerebrospinal fluid, a variety of pneumococcal components, particularly cell wall components, incite the inflammatory response (Todar, 2003).
3.10 Cultivation
Streptococcus pneumoniae is fastidious bacterium, growing best in 5% carbon dioxide. In all cases, growth requires a source of catalase (e.g. blood) to neutralize the large amount of hydrogen peroxide produced by the bacteria. In complex media containing blood, at 37ºC, the bacterium has a doubling time of 20-30 minutes (Todar, 2003).
S pneumoniae is fermentative aerotolerant anaerobe. It is usually cultured in media that contain blood. On blood agar, colonies characteristically produce a zone of alpha (green) hemolysis, which differentiates S pneumoniae from the group A (beta hemolytic) streptococcus, but not from commensal alpha hemolytic (viridans) streptococci which are co-inhabitants of the upper respiratory tract. Special tests such as bile solubility, optochin sensitivity must be routinely employed to differentiate the pneumococcus from Streptococcus viridans (Todar, 2003).
S pneumoniae is a very fragile bacterium and contains within itself the enzymatic ability to disrupt and to disintegrate the cells. The enzyme is called an autolysin. The physiological role of this autolysin is to cause the culture to undergo a characteristic autolysis that kills the entire culture when grown to stationary phase. Virtually all clinical isolates of pneumococci harbor this autolysin and undergo lysis usually beginning between 18-24 hours after initiation of growth under optimal conditions. Autolysis is consistent with changes in colony morphology. Colonies initially appear with plateau type morphology, and then start to collapse in the centers when autolysis begins (Todar, 2003).
3.11 Identification
Streptococcus pneumoniae is Gram positive elongated diplococcus but may also occur singly and in short chains. Individual cells are between 0.5 and 1.25 micrometer in diameter (Todar, 2003). This pneumococcus is non motile, non sporing and capsulated (non capsulated following culture). In Gram stained smears from specimens, the capsule can often be detected as an unstained empty area around the diplococcus (Kaijalainen, 2006).
Optimal growth occurs in an increased carbon dioxide atmosphere. On blood agar, S pneumoniae forms translucent or mucoid colonies, 1-2 mm in diameter following overnight incubation at 35-37ºC. In young culture, the colonies are raised but as the culture ages, the colonies become flattened, with a depressed central part and raised edges giving them a ringed appearance (draughtsmen colony). The pneumococcus shows alpha hemolysis i.e., colonies are surrounded by an area of partial haemolysis with a greenish discoloration in the medium (Cheesbrough, 2005).
The minimum criteria for the identification and distinction of pneumococci from other streptococci are bile solubility, optochin sensitivity, gram positive staining and α-hemolytic activity. Pneumococci cause alpha hemolysis on agar containing sheep blood. Under anaerobic conditions they switch to beta hemolysis caused by an oxygen labile hemolysin. Typically, pneumococci form a 14 mm zone of inhibition around a 5 µg Optochin disc, and undergo lysis by bile salts. This lysis depends upon the presence of an autolysin enzyme, LytA (Kaijalainen, 2006).
3.12 Distribution of pneumococcal serotypes
Distribution of pneumococcal serotypes associated with disease can vary according to several parameters, including geographic area, period of analysis, and age group (Teixeira, 2002). Results of surveillance in more industrialized countries suggest that serotype distribution associated with infection in children is different from that observed in adults.
Among the 90 different polysaccharide serotypes, some are more virulent than others. A small number of them, approximately 10 serotypes, are common in pneumococcal infections. Serotypes 1, 3, 4, 6, 7, 9, 14, 18, 19, and 23 are the most frequent in children’s disease. According to a recent review, serotypes 1, 3, 5, 6, 14, 19 and 23 are comprehensive types in invasive pneumococcal infections on several continents (Kaijalainen, 2006).
Information on the regional distribution of pneumococcal serotypes is essential for the development and use of appropriate pneumococcal vaccines in developing countries. Serotypes 1, 5, 7, 19, and 23 are commonly encountered in India (Siberry et al., 2001).
Determining the serotypes of S pneumoniae from different clinical specimens is important as the vaccine production is based on the most common serotypes (Ozalp et al., 2004).
Effective surveillance of pneumococcal disease and its serotypes is needed to accurately map the magnitude of the problem and help evaluate the impact of available vaccines (www.infectioncontroltoday.com).
Fortunately, new vaccines to prevent deadly pneumococcal infections are now available and widely used in many countries in North America and Europe. With systematic surveillance in place and a coordinated effort to introduce pneumococcal vaccines we could save millions of children’s lives and make a significant move towards meeting a key U.N. Millennium Development Goal of reducing child mortality by two-thirds by 2015 (www.infectioncontroltoday.com).
3.13 Genetics
S pneumoniae has a natural transformation system as a mechanism for genetic exchange. This process is of medical significance because it clearly underlies the explosion of antibiotic resistance in the bacterium over the past 20 years. For example, penicillin resistance is due to altered penicillin binding proteins (PBPs) which exhibit a low affinity for beta lactam antibiotics. Comparison of the nucleotide sequences encoding the PBPs in S pneumoniae and S mitis demonstrates that horizontal gene transfer has occurred between these two bacteria. In the upper respiratory tract of the host, horizontal exchange of genetic information could take place between strains of pneumococci that co-habitat or compete for dominance as normal flora (Todar, 2003).
S pneumoniae can also develop antibiotic resistance by the timeless process of mutation and selection. The bacterium has a relatively fast growth rate and achieves large cell densities in an infectious setting. These conditions not only favor the occurrence of natural transformation, but also the emergence of spontaneous mutants resistant to the antibiotic (Todar, 2003).
3.14 Antimicrobial susceptibility
Antibiotics with activity against pneumococci include Penicillin, Erythromycin, Co-trimoxazole, and Cephotaxime. Isolates should also be tested for sensitivity to Tetracycline, Chloramphenicol (Cheesbrough, 2005).
Penicillin resistant strains are becoming an increasing problem in tropical Africa, South Africa, and else where. At the present time, clinical laboratories are advised to screen all clinical isolates of S pneumoniae for penicillin resistance. Such screening can be best performed by the use of a disc containing 1 µg of Oxacillin (WHO, 1994).
Antibiotic-resistant S. pneumoniae peaks at 0-2 years: resistance to penicillin, erythromycin.(Infectious Diseases)(Streptococcus pneumoniae): An article from: Pediatric News
3.15 Antimicrobial resistance
Also of concern, is the increased emergence of antibiotic resistance, especially in the past decade. Multiple antibiotic resistant strains of S pneumoniae that emerged in the early 1970s in Papua New Guinea and South Africa were thought to be a fluke, but multiple antibiotic resistance now covers the globe and has rapidly increased since 1995 (Todar, 2003).
Resistance to penicillin in S pneumoniae is increasing throughout the world. The problem is particularly common in Spain, Eastern Europe, South Africa, South America, New Guinea and Korea where resistance up to 30-50% is commonly reported (Catterall, 1999).
Increases in penicillin resistance have been followed by resistance to cephalosporins and multidrug resistance. The incidence of resistance to penicillin increased from <0.02 in 1987 to 3% in 1994 to 30% in some communities in the united states and 80% in regions of some other countries in 1998. Resistance to other antibiotics has emerged simultaneously: 26% resistant to Trimethoprim-sulphamethoxazole, 9% resistant to cefotaxime, 30% resistant to macrolides, and 25% resistant to multi drugs. Resistant organisms remain fully virulent but seem to have arisen in less than 10 serotypes. Serotypes 6A, 6B, 9V, 14, 19A and 23F are included in the vast majority of resistant strains (Todar, 2003). While antimicrobial resistance spawned by indiscriminate antibiotic usage in developing countries has received much attention, less understood are the epidemiology of antibiotic resistance and factors contributing to regional differences. Prevalence rates of resistance among nasopharyngeal or blood stream isolates of S pneumoniae and H influenzae from children in developing countries have been recently reviewed. The majority of S pneumoniae in South Asia are now Cotrimoxazole resistant- raising the question of whether W.H.O. ARI program should shift from Cotrimoxazole to more expensive Amoxicillin for treatment (Zaidi, 2003). Penicillin resistance among pneumococcal isolates in South Asia has also emerged and is gradually increasing, with 5-10% of isolates currently resistant. In Pakistan, Cotrimoxazole therapy has increasingly failed; two studies have found Cotrimoxazole to be ineffective in one-third of patients with pneumonia; and children under age of 1 year were especially susceptible to treatment failure (Zaidi, 2003). Comprehensive surveillance of drug resistance patterns in this microorganism is needed to guide prevention and control efforts. 3.16 Prevention of pneumococcal infection The increasing rate of antibiotic resistance in S pneumoniae complicates the elimination of pneumococci by therapy and strongly supports the application of new vaccine strategies. Preventive strategies for pneumococcal infection include use of the 23-valent polysaccharide pneumococcal vaccine for individuals older than 2 years of age, and routine immunization of infants and children with the 7-valent polysaccharide-protein conjugate pneumococcal vaccine. As vaccine coverage (i.e. immunization rate) in children and adults increase, the disease burden of invasive pneumococcal infections decreases (Weber and Rutala, 2003). 3.16.1 Anti-pneumococcal vaccine In 2002, 11 million infants living in developing countries died from vaccine preventable diseases. Pneumococcal infection accounts for more deaths than any other vaccine-preventable bacterial disease. Those most commonly at risk for pneumococcal infection are children between 6 months and 4 years of age and adults over 60 years of age (Dejsirilert et al., 1999). Capsular polysaccharide is the basis of the current anti pneumococcal vaccine. New vaccines to prevent deadly pneumococcal infections are now available and widely used in many countries in North America and Europe (www.infectioncontroltoday.com). Polysaccharide (PS) vaccine Given the 90 different capsular types of pneumococci, a comprehensive vaccine based on polysaccharide alone is not feasible. Thus, vaccines based on a subgroup of highly prevalent types have been formulated (Todar, 2003). The number of serotypes in the vaccine has increased from four in 1945, to 14 in the 1970s, and finally to the current 23-valent (polysaccharide vaccine) formulation (one dose 0.5 ml of the 23-valent vaccine contains 25 µg capsular polysaccharide antigen of each serotypes 1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F) (www.cdc.com, 1998). These serotypes represent 85%-90% of those that cause invasive disease in western industrialized countries and the vaccine efficacy is estimated at 60%. However, underutilization of the vaccine is so extensive that the pneumococcus remains the most common infectious agent leading to hospitalization in all age groups. This is further complicated by the fact that polysaccharides are not immunogenic in children under the age of 2 years where a significant amount of disease occurs (Todar, 2003). The currently available pneumococcal vaccines are manufactured by both Merck and Company, Inc. (Pneumovax 23) and Lederle Laboratories (Pnu-Imune 23). These vaccines were licensed in the United States in 1983 (www.cdc.com, 1998). Protein-polysaccharide (Conjugate) vaccine Over the past 15 years, several vaccine manufacturers have developed pneumococcal conjugate vaccines in which a number of S pneumoniae PS are covalently coupled to a protein carrier. Conjugate vaccines elicit higher antibody levels and a more efficient immune response in infants, young children and immunodeficient persons than the PS vaccines. Moreover, these vaccines suppress nasopharyngeal carriage of the pathogen and reduce bacterial transmission in the community through herd immunity, which adds considerable value to their implementation. Conjugate vaccines immunization followed by PS vaccine boosting might provide a foundation for lifelong protection against pneumococcal disease (WHO, 2006). Furthermore, the world wide increase in penicillin resistance among pneumococci and the limited use of the pneumococcal vaccine suggest that morbidity and mortality from pneumococcal disease may increase. CHAPTER IV 4 Materials and Methods 4.1 Materials Details of the materials used during the study are provided in Annex II, III, IV and V. 4.1.1 Equipments Biological Safety Cabinet Dalton Incubator Sakura: IF-3B, Tiyoda Manufacturing Company, Japan. Autoclave Sakura Neoclave ASV 2402, Tiyoda Hot Air Oven Sakura Clinioven TF-21 Microscope Olympus, PM-10 ADS; Olympus Optical Co. Ltd., Japan. Water Distillation Plant Adventec. GS-200, Japan. Weighing Machine Chyo JL- 200; Chyo Balance Co. Ltd., Japan. Refrigerator Toshiba Company, Japan Anaerobic Gas Jar Oxoid 4.2 Methodology 4.2.1 Study design The study was hospital based prospective study for which nasopharyngeal swab specimens were collected from the children attending OPD of KCH and all the processing were conducted in Health Research Laboratory, IOM, TUTH. All together 188 children attending KCH between February 2007 to September 2007 were involved. Consent was taken from guardian of every child and also from the child whenever appropriate or possible prior to enrollment. All the guardians were asked for the information as specified in the prepared questionnaire. 4.2.2 Sample collection After obtaining consent, nasopharyngeal swab specimen was collected using specifically designed pediatric sized swab of thin flexible aluminum shaft tipped with Dacron polyester. The flexible swab was inserted through one nasal aperture into the posterior wall of the nasopharynx. Once the swab was in place, it was slightly rotated and left for about 10 seconds to saturate the tip and removed slowly. Once a swab specimen was collected, it was placed in a tube of STGG medium. The excess wire handle was cut off from the swab using scissors sterilized with an alcohol wipe leaving the swab itself in the medium. The specimens were processed within 3 hours of collection following standard laboratory procedures. 4.2.3 Culture In the laboratory, the specimens were vortexed for 20-30 seconds to disperse organisms from the swab tip. Each specimen was inoculated on blood agar plate with 7% sheep blood. The inoculated plates were then incubated overnight at 37º C in CO2 enriched atmosphere. 4.2.4 Examination of culture plate Culture plates after overnight incubation were looked for the growth of S pneumoniae characterized by small, grayish mucoid colonies surrounded by greenish zone of hemolysis. One or two typical colony was picked and sub-cultured on blood agar plate with 7% sheep blood to obtain pure culture for further processing. 4.2.5 Identification of S pneumoniae The isolates were identified following Bergey’s Manual of Systematic Bacteriology. Colony of S pneumoniae appeared as small, grayish mucoid surrounded by greenish zone of haemolysis. Young colonies appeared raised while as the culture aged; colony became flattened at center with raised edges. On Gram staining, they appeared Gram positive elongated diplococcus and some occurred singly and in short chain under microscope. S pneumoniae was further confirmed by sub-culturing the isolate on BA and placing an Optochin disc (5 µg) on the primary streak line. 14 mm zone of inhibition around the disc after incubation at 37ºC for 24 hours confirmed S pneumoniae. It was also bile soluble. 4.2.6 Antibiotic susceptibility test Antibiotic susceptibility test of the isolated pneumococci was performed by modified Kirby-Bauer Disc Diffusion Technique. For the test, a suspension of pure overnight culture of the organism was prepared in Mueller-Hinton broth with its turbidity equivalent to 0.5 McFarland standard and it was swabbed on entire surface of Mueller-Hinton Agar plate with 7% sheep blood using sterile cotton swab. 6 antibiotic discs were placed around the edge of the plate and incubated overnight at 37ºC in CO2 enriched atmosphere. Diameter of zone of inhibition around each disc was measured and interpreted according to NCCLS recommendation. Zone size interpretative chart is given in Appendix VII. 4.2.7 Serotyping Serotyping of the isolated pneumococci was done by coagglutination method using Pneumotest (pneumococcal antisera) kit. The Pneumotest kit contains 12 pooled sera (A to F plus H, and P to T). Capsular polysaccharide of pneumococci is the basis of this serotyping technique. It is based on capsular reaction due to interaction between pneumococcal capsular polysaccharide and its homologous antibody. A positive reaction is indicated by the bacterial coagglutination as a result of an in situ immunoprecipitation. The procedures of serotyping and interpretation of result are given in Appendix VIII and IX. 4.2.8 MIC testing Minimum Inhibitory Concentration (MIC) of Oxacillin resistant isolates was determined against penicillin by E-test. The procedure of determining MIC by E-test is given in Appendix X. 4.2.9 Quality control It is of utmost importance to perform quality control throughout the study to obtain results that are both reliable and desirable. So, quality control was applied at different level during the study. • Samples were processed on the same day of collection as soon as possible. • All the processing of samples was performed aseptically within the biological safety cabinet. • Freshly prepared media were used every time and each batch of media was put into quality check. For that a freshly prepared media plate was incubated with ATCC 49619 and another was incubated uninoculated. Growth obtained on the inoculated plate helped to make out the type of growth to look for whereas absence of growth on uninoculated plate clearly indicated that the media was sterile. In this way, ATCC 49619 was used as control strain for both identification and antibiotic susceptibility test. CHAPTER V 5 Result Nasopharyngeal swab specimens from 188 children attending OPD of KCH were obtained randomly after obtaining informed consent from their parents. The collected samples were processed in the Health Research Laboratory, IOM, TU. The findings of the study were compiled and then analyzed with the chi-square statistic. 5.1 Pattern of result Of all 188 specimens investigated for S pneumoniae, 65 (34.57%) of them showed the growth of S pneumoniae and 123 (65.43%) were S pneumoniae negative as shown below in Figure 1. Figure 1: Subjects enrolled and gender wise distribution of S pneumoniae among them 5.2 Gender wise distribution of S pneumoniae Out of 65 positive cases, 33 (50.77%) were male and 32 (49.23%) were female. Gender wise distribution of nasopharyngeal carriage of S pneumoniae was statistically not significant (P > 0.05) (Table 1).
Table 1: Gender wise distribution of S pneumoniae
Gender Number Percentage
Male 33 50.77%
Female 32 49.23%
Total 65 100%
5.3 Prevalence of S pneumoniae colonization in different age groups
Nasopharyngeal carriage rate was highest among children of age group 2 to 12 months followed by age group 37 to 48 months, 13 to 24 months, 49 to 60 months and 25 to 36 months. The result showed that nasopharyngeal carriage of S pneumoniae was statistically significant (P < 0.05) among children of age group 2 to 12 months (Table 2). Table 2: Age wise distribution of S pneumoniae Age (in months) Number Percentage 2-12 34 52.31% 13-24 8 12.31% 25-36 5 7.69% 37-48 11 16.92% 49-60 7 10.77% Total 65 100% 5.4 Distribution of S pneumoniae in relation to type of cooking stove Nasopharyngeal carriage rate of S pneumoniae was highest among the children from the family using firewood cooking stove 47.69% followed by kerosene cooking stove 29.23% and gas cooking stove 23.08%. The association between type of cooking stove and carriage is statistically significant (P < 0.05) (Table 3). Table 3: Distribution of S pneumoniae in relation to type of cooking stove Type of cooking stove Number Percentage Firewood 31 47.69% Kerosene 19 29.23% Gas 15 23.08% Total 65 100% 5.5 Distribution of S pneumoniae according to family type Nasopharyngeal carriage rate of S pneumoniae was found to be highest among children from extended family (50.77%) in comparison to children from nuclear family (49.23%) however, it was not statistically significant (P > 0.05) (Table 4).
Table 4: Distribution of S pneumoniae according to family type
Family type Number Percentage
Extended 33 50.77%
Nuclear 32 49.23%
Total 65 100%
5.6 Distribution of S pneumoniae in relation to the presence of other children in the family
Nasopharyngeal carriage rate of S pneumoniae was same in children from family where there was no other child and where there was only one other child accounting for 33.85% each. Likewise, the carriage rate was 32.3% in children from family where number of other children was equal to or greater than 2. The association between number of other children and carriage rate was statistically significant (P < 0.05) (Table 5). Table 5: Distribution of S pneumoniae in relation to the presence of other children in the family Number of other child in the family Number Percentage 0 22 33.85% 1 22 33.85% ≥2 21 32.3% Total 65 100% 5.7 Distribution of different serotypes of the isolated S pneumoniae All S pneumoniae isolates were serotyped by coagglutination method using Pneumotest (pneumococcal antisera) kit. Among 65 S pneumoniae isolates, 5 were Non-Typeable (NT). Remaining 60 isolates were found to belong to 16 different serotypes. Frequency of occurrence of different serotypes is given in descending order: serotype 19 and serotype 6: 18% each, serotype 15: 9%, serotype 14: 8%, serotype 23 and serotype 11: 6% each, serotype 20, serotype 7 and serotype 3: 5% each, serotype 12: 3% and serotype 18, serotype 17, serotype 10, serotype 9, serotype 8 and serotype 5: 2% each (Table 6). Table 6: Distribution of different serotypes of the isolated S pneumoniae Serotypes Number Percentage 19 12 18% 6 12 18% 15 6 9% 14 5 8% 23 4 6% 11 4 6% 20 3 5% 7 3 5% 3 3 5% 12 2 3% 18 1 2% 17 1 2% 10 1 2% 9 1 2% 8 1 2% 5 1 2% Non-Typeable 5 8% Total 65 100% 5.8 Antimicrobial susceptibility pattern of S pneumoniae All 65 S pneumoniae isolates were tested against Cefotaxime, Cotrimoxazole, Chloramphenicol, Erythromycin, Tetracycline and Oxacillin using modified Kirby-Bauer disc diffusion method. Of them, 100% of these isolates were susceptible to Cephotaxime and Chloramphenicol, 98.46% were susceptible to Erythromycin. 78.46% were susceptible to Tetracycline and 12.31% were moderately susceptible to Tetracycline. On the other hand, 49.23% and 10.77% were susceptible and moderately susceptible respectively to Cotrimoxazole. 6.15% were found to be resistant to Oxacillin. Table 7: Antimicrobial susceptibility pattern of S pneumoniae Antibiotic Susceptible Moderately susceptible Resistant Total Number Percentage Number Percentage Number Percentage Number Percentage Cephotaxime 65 100% 0 0 0 0 65 100% Chloramphenicol 65 100% - - 0 0 65 100% Cotrimoxazole 32 49.23% 7 10.77% 26 40% 65 100% Erythromycin 64 98.46% 0 0 1 1.54% 65 100% Tetracycline 51 78.46% 8 12.31% 6 9.23% 65 100% Oxacillin 55 84.62% - - 10 15.38% 65 100% 5.9 MIC testing Oxacillin resistant isolates were tested against Penicillin using E-test strip. MIC of Penicillin of two Oxacillin resistant isolates was found moderately susceptible while that of remaining eight Oxacillin resistant isolates was found susceptible. CHAPTER VI 6 Discussion and conclusion 6.1 Discussion Many of the bacteria that cause ARI can be isolated as a part of the normal flora of healthy people. Streptococcus pneumoniae is a major community acquired pathogen responsible for vast majority of diseases particularly in young children below 5 years of age. Bacterial colonization of nasopharynx starts immediately after birth and continues throughout life with small changes and S pneumoniae also belongs to normal flora which is easily transmitted. Under certain circumstances, these colonizing microorganisms go on to cause disease. In this study, 188 nasopharyngeal swab specimens were collected from children attending OPD of KCH to determine colonization of nasopharynx by S pneumoniae. Of the total 188 nasopharyngeal swab specimens processed, only 65 (34.57%) of them showed the growth of S pneumoniae. This is supported by the study carried out by Todar in 2003, in which, nasopharyngeal colonization with pneumococci occurred in 40% of the cases. Among 65 subjects positive for S pneumoniae colonization, 33 (50.77%) were male and 32 (49.23%) were female. This showed that there was no correlation between gender and nasopharyngeal carriage (P > 0.05). This may be because, unlike in case of adults where males are more prone to ARI due to mobility and other predisposing factors such as smoking, children below 5 years of age stays at home irrespective of their gender. This can be correlated with findings of Malla et al., (2005) who reported similar results: 49% in female and 51% in male.
Nasopharyngeal carriage rate was found to be highest among children of age group 2-12 months accounting for 52.31%. The prevalence rate was found to be 16.92%, 12.31%, 10.77% and 7.69% in children of age group 37-48, 13-24, 49-60 and 25-36, respectively. The result of this study suggested a strong correlation between age of children and nasopharyngeal colonization with pneumococci (P < 0.05). High prevalence of pneumococci carriage in children belonging to 2-12 months age group is mainly due to close and attached mother to child relationship. Children of this age group are more in contact with their mother than children of other age group. As such, children of this age group mostly acquire their normal flora from their own mother according to the results of the study carried out by Catterall (1999). The carriage rate decreased with increase in age of child up to 36 months. It is attributable to decreased child to mother relationship with increase in age of child in comparison to 2 to 12 month’s age group. Children from 2 to 24 months of age represent preschool children and children attending child day care centers and nurseries among which carriage rates are highest. Children of age group 2 to 24 months contributed to highest carriage rate which was 64.62%. Carriage rate amongst children from 37 from 60 months of age is dependent on likelihood of their contact with other children according to Catterall (1999). Nasopharyngeal carriage of S pneumoniae was highest, i.e. 47.69% among the children from family using firewood cooking stove followed by kerosene cooking stove 29.23% and gas cooking stove 23.08%. This clearly reflected strong correlation between carriage rate and type of cooking stove which is also statistically significant (P < 0.05). Cooking with firewood stove results in highest level of indoor air pollution followed by kerosene stove and gas stove. Smoke in the home from cooking stove is also an important contributing factor of nasopharyngeal colonization. Women and children are predominantly the victims of smoke produced in kitchen while cooking. Women spend at least three hours a day in kitchen and children under five years of age spend most of the time with their mothers. So they are also exposed to smoke (Malla et al., 2005). Indoor pollution has considerable impact on pneumococcal carriage and its rates according to Cant et al, (2002) and Kaijalainen (2006). The respiratory tract is directly exposed to many potential pathogens via smoke, soot and dust that are inhaled from the air as stated by WHO (2003). Family type is also considered in this study because its type represents the number of family members to whom the child in the family is exposed. Extended family has large number of family members whereas nuclear family has few family members. In this study, carriage rate of S pneumoniae was found to be 50.77% in children from extended family and 49.23% from nuclear family. Here, although carriage rate was highest in children from extended family, there was no significant difference in carriage rate between children from extended and nuclear family. The most probable reason behind it may be because the children under 5 years of age spend most of their time with their mothers rather than with other members of the family. From this, we can conclude that there is no correlation between nasopharyngeal carriage and family type (P > 0.05).
Although carriage of S pneumoniae was very similar among families where there were no other children, where there were only one child and where there were two and more than two children, there is association between the carriage and number of other children in the family. These are also associated statistically (P < 0.05).
In the family where there was no other child, obviously the child gets greater chance to be with its mother. In the family where there is another one child, due to less age difference between two, both of them have nearly equal chance to spend with their mother. So carriage was similar. In the family where there are two and more other children, the carriage rate was higher. It may be due to transmission of pneumococci among children.
The nasopharyngeal isolates of S pneumoniae were serotyped by coagglutination method using Pneumotest (pneumococcal antisera) kit. The isolates were found to belong to 16 different serotypes. Frequency of occurrence of different serotypes is given in descending order: serotype 19 and serotype 6: 18% each, serotype 15: 9%, serotype 14 8%, serotype 23 and serotype 11: 6% each, serotype 20, serotype 7, and serotype 3: 5% each, serotype 12: 3% and serotype 18, serotype 17, serotype 10, serotype 9, serotype 8 and serotype 5: 2% each. Remaining 8% were found to be Non-Typeable.
Information on the regional distribution of pneumococcal serotype is essential for the development and use of appropriate pneumococcal vaccine in developing countries. Determining the serotype of S pneumoniae from different clinical specimens is important as the vaccine production is based on the most common serotypes (Siberry et al., 2001 and Ozalp et al., 2004).
Serotype 1 is regarded as most invasive. However, this serotype was not encountered in this study. Serotypes 1, 3, 5, 6, 14, 19, and 23 are considered comprehensive types in invasive pneumococcal infections (Kaiijalainen, 2006). Except serotype 1, all serotypes considered comprehensive type in invasive pneumococcal infections were found in the study.
Serotypes 19 and 6 were found to be the most common serotypes in present study. Other common serotypes included 15, 14, 23, 11, 20, 7, 3 and 12.
Serotypes 19, 6, 15, 23, 9, 11, 8, 7, 17, 20, 22 were commonly involved in nasopharyngeal colonization in children according to the report by Ozalp et al., (2004). Serotypes 3, 19, 23, 6, 14 were most common nasopharyngeal isolates in children in the study carried out by Marchisio et al., in 2002.
All the isolated pneumococci were found to be susceptible to Cephotaxime and Chloramphenicol. Erythromycin is also effective drug of choice since only 1.54% was found to be resistant to it. Tetracycline can also be used for therapy since only 9.23% isolates were found to be resistant to it.
Cotrimoxazole was recommended by W.H.O. to treat against infection caused by S pneumoniae due to its lesser side effects, lower cost and easy availability. However, in the current study, Cotrimoxazole showed lowest susceptibility with 40% resistance. Also in a similar study, previously carried out by Malla et al., in 2005, it was the least effective drug against S pneumoniae. In Pakistan, two studies have found Cotrimoxazole to be ineffective in one third of patients with pneumonia; and children under age of 1 year were especially susceptible to treatment failure. The majority of S pneumoniae in South Asia are now Cotrimoxazole resistant- raising the question of whether W.H.O. should shift from Cotrimioxazole to more expensive Amoxicillin for treatment (Zaidi, 2003).
The key factor behind the emergence of Cotrimoxazole resistant pneumococci is unnecessary use of antibiotic for viral respiratory infections because of widespread confusion over the difference between viral and bacterial respiratory infections. Also, antibiotics are freely available in the market without prescriptions. Cotrimoxazole is the cheapest antibiotic which is widely available in the market in comparison to other antibiotics and it is most commonly used by people without discrimination between viral and bacterial respiratory infections.
In the current study 15.38% of the isolates were found to be Oxacillin resistant. In study carried out by Malla et al., in 2005, 5.12% of nasopharyngeal isolates of pneumococci were found to be Oxacillin resistant. There has been a slight increase, in this study, in Oxacillin resistance. Clinical laboratories are advised to screen all important isolates of S pneumoniae for Penicillin resistance. Therefore, though Oxacillin is not used in therapy, it is used in vitro antimicrobial susceptibility testing of S pneumoniae for predicting resistance of S pneumoniae to Penicillin because of its greater resistance to deterioration during its storage and it provides the most reproducible results.
On performing E-test of Oxacillin resistant strains against Penicillin, MIC of two of the Oxacillin resistant pneumococcal isolates was found moderately susceptible and remaining was found to be susceptible to Penicillin.
Though Oxacillin resistance rate is not large, the value is alarming. Though disc sensitivity testing accurately reflects whether an organism is resistant to most antimicrobial agents, disc testing of Oxacillin resistance for S pneumoniae is not sufficient to distinguish between complete and partial resistance since it does not distinguish penicillin intermediate resistant strains from strains that are penicillin resistant. MIC testing of isolates identified by disc as resistant is needed to quantify the level of resistance of S pneumoniae to Penicillin. The higher the MIC, the more likely treatment is to be ineffective (WHO). Therefore MIC of Oxacillin resistant isolates was determined by E-test against Penicillin.
Penicillin resistance among pneumococcal isolates in South Asia has also emerged and is gradually increasing, with 5-10% of isolates currently resistant. In Korea, it is 25-30% (Zaidi, 2003).
6.2 Conclusion
Nasopharyngeal carriage study of Streptococcus pneumoniae was carried out among children attending Kanti Children’s Hospital and the carriage rate was found to be approximately 35%.
CHAPTER VII
7 Summary and Recommendation
7.1 Summary
1. Nasopharyngeal colonization with S pneumoniae was found in approximately 35% of the studied population and colonization rate was similar in both male and female.
2. The colonization rate was greater in the children from the family using firewood as cooking stove.
3. Children of age group 2 to 24 months contributed to highest carriage rate.
4. Among the isolated pneumococci, 5 were found to be Non-Typeable and remaining were found to belong to 16 different serotypes.
5. On performing antibiotic susceptibility test, Cephotaxime, Chloramphenicol, Erythromycins were found to be most effective antibiotics against the isolates of pneumococcus whereas Cotrimoxazole showed least susceptibility.
6. On performing MIC testing of the Oxacillin resistant isolates by E-test, two of the isolates were found to be moderately susceptible to Penicillin and remaining eight isolates were found susceptible. From this, we could conclude that Penicillin resistance had not evolved in the isolates of S pneumoniae in children.
7.2 Recommendations
Following recommendations are made based on this study for further study:
1. Current study was hospital based study. This study can also be carried out in community and also in children attending nurseries and kindergarten as a comparative study among themselves and between two groups.
2. The study on seasonal variation and impact of season on nasopharyngeal colonization can also be carried out.
3. It is also possible to determine MIC of Cotrimoxazole resistant S pneumoniae as recommended by WHO to quantify the level of resistance as disc testing of Cotrimoxazole resistance for S pneumoniae isolates is not sufficient to distinguish between complete and partial resistance.
4. It is also possible to compare nasopharyngeal colonization of mother and child with pneumococci and accessing epidemiology in the two.
CHAPTER VIII
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