Prevalence And Rapid Diagnosis Of Acute Bacterial Meningitis In Childhood In Bangladesh
1. INTRODUCTION AND REVIEW OF LITERATURE
1.1 INTRODUCTION
Meningitis is an inflammation of the membranes and cerebrospinal fluid (CSF) that encases and bathes the brain and spinal cord. Meningitis is a serious disease that includes several types. These include bacterial meningitis, acute bacterial meningitis, viral meningitis, aseptic meningitis and chronic meningitis.
Meningitis is a serious disease that can be life-threatening and result in permanent complications, such as coma, shock, and death. Meningitis is a serious infection of one of the membranes that surrounds the brain. Acute meningitis caused by bacteria is called acute bacterial meningitis and develops very quickly in a matter of hours or days. Acute bacterial meningitis is generally the most serious type of meningitis.
The pathogens that can cause many forms of meningitis are carried by humans in the nose and throat and are spread into the air by coughing and/or sneezing. Once pathogens are airborne, they can be picked up by anyone who breathes them into their respiratory tract. The pathogens then spread from the respiratory tract to the blood stream and to the nervous system.
Symptoms of meningitis include a high fever and stiff neck. Serious complications can occur, especially with acute bacterial meningitis. In some cases death can happen in a matter of days
Making a rapid and accurate diagnosis of meningitis begins with taking a thorough personal and family medical history, including symptoms, and completing a physical examination. Diagnostic tests include a lumbar puncture, also called a spinal tap, which involves withdrawing a small sample of cerebrospinal fluid (CSF) from the spine with a needle. The sample of CSF is tested for white blood cells and other indications of meningitis.
Additional tests may be performed in order to rule out or confirm other diseases that may accompany meningitis or cause similar symptoms, such as high fever, headache, and neck stiffness. These may include a throat culture, CT, or X-rays.
It is possible that a diagnosis of meningitis can be missed or delayed because some symptoms, such as fever, headache, and nausea and vomiting, are similar to symptoms of other diseases.
In infants, signs of acute bacterial meningitis can include excessive crying, excessive sleepiness, difficulty with feeding, and a bulging of the soft spot on the top of the head. Serious complications of acute bacterial meningitis include kidney failure and permanent neurological damage, such as blindness, hearing loss, brain damage, and paralysis.
? Causes of bacterial meningitis:
? other causes of bacterial meningitis mainly in newborns:
Pseudomonas (type of Nosocomial infections)
Acute bacterial meningitis is one of the common causes of morbidity and mortality in children under 5 years of age in developing countries (Saha, 1997). It is well known that H. influenzae and S. pneumoniae and N. meningitidis are responsible for 80% of meningitis cases (Chowdhury et al., 1992).
1.2 MENINGITIS CAUSES: RISK FACTORS
The following conditions have been cited in various sources as potentially causal risk factors related to Meningitis:
Contacts of confirmed meningococcal cases
Overseas travel – especially to Africa or parts of Asia where meningococcus is more common.
Institutions – have been associated with meningococcal disease outbreaks
More risk factors.
Data on the aetiology of acute bacterial meningitis in Bangladesh are few. This study has been designed to shed light on the incidence, rapid diagnosis and aetiological trends in bacterial meningitis in childhood less than twelve years of age in Bangladesh.
1.3 BACTERIAL MENINGITIS
The subarachnoid space and its CSF are relatively defenseless in stopping invasion by bacterial pathogens because of the CSF’s paucity of phagocytic cells and low concentrations of complement and immunoglobulin. Unchecked invasion and multiplication of bacteria in the CSF result in meningitis. The pathophysiology of bacterial meningitis has been studied experimentally and is reasonably well understood (McGee et al., 1990, Saez-Llorens et al., 1991 and Tunkel et al., 1990). Inflammation of the meninges is initiated by the presence of bacterial lipopolysaccharide, teichoic acid, and/or other bacterial cell wall components in the subarachnoid space. The bacterial antigens stimulate monocytes to produce the cytokine interleukin-1 and stimulate macrophages, astrocytes, microglial cells, ependymal cells, and endothelial cells in the central nervous system to produce the cytokine tumor necrosis factor (cachectin). Tumor necrosis factor and interleukin-1 probably act synergistically to elicit inflammatory responses which manifest clinically as meningitis. A logical temporal sequence of such responses is as follows: chemotaxis and adherence of polymorphonuclear leukocytes to cerebral capillaries; damage to capillary endothelial cells; structural changes in the bloodbrain barrier; cytotoxic parenchymal edema; increased intracranial pressure; decreased intracranial perfusion; cerebral infarction; and focal or diffuse brain damage.
Acute bacterial meningitis is a severe childhood illness. In developing countries like Bangladesh, it is a leading cause of bacterial meningitis (Salisbury, 1998), responsible for over 200,000 cases and more than 40,000 deaths annually (Mulholland et al., 1997; Salisbury, 1998). Haemophilus influenzae, Neisseria meningitidis and Streptococcus pneumoniae remain important pathogens (Schlech et al., 1985, Schuchat et al., 1997).
The prevalence of these organisms varies from place to place, by age and by season (Schlech et al., 1985), but N. meningitidis is more often the commonest cause of meningeal infection (Bell & Silber, 1971) with S. pneumoniae (Spink & Su, 1960) and H. influenzae (McGowan, 1974) being second and third respectively. However, the order is reverse in several studies in Bangladesh (Saha et al., 1997). Recurring epidemics of meningococcal disease (Booy & Kroll, 1998), increased antibiotic resistance among pneumococci (Goldstein & Acar, 1996), and failure to introduce conjugate Hib vaccines into many developing countries means that bacterial meningitis remains a serious global health problem.
Children are most likely to get meningitis during their first year (Schlech et al., 1985; Panjarathinam & Shah, 1993). Children who were infected as neonates had more health and developmental problems than those who had meningitis when they were older than one month (Bedford et al., 2001). The rates of severe or moderate disability differ widely between children infected with different organisms (Bedford et al., 2001). Infection with S. pneumoniae has been reported to be associated with a higher rate of disability than infection with H. influenzae and N. meningitides (Bedford et al., 2001; Baraff et al., 1993).
Even with the advances in the development of many powerful antimicrobial agents, bacterial meningitis still remains a serious cause of morbidity and mortality in childhood. Although it is widely acknowledged that the consequences of meningitis in infancy may be severe, there are few studies that focus on the etiologic diagnosis of bacterial meningitis (Rahman et al., 1990; Saha et al., 1997; Saha 2003; Saha et al., 2005; Hoque et al., 2006), and no reliable data from large prospective studies that focus on the outcome of infection in infancy or fatality in Bangladesh. Since meningitis is potentially hazardous disease of childhood, diagnostic tests that are readily available, easy to interpret and simple to perform are of paramount importance. Lumbar puncture is frequently performed in primary care (Whistle et al., 1995; Visser & Hall, 1980). Commonly performed tests on cerebrospinal fluid (CSF) include protein and glucose levels, cell counts and differential, microscopic examination, and culture (Seehusen et al., 2003). Culture is the “gold standard” for determining the causative organism in meningitis (Seehusen et al., 2003). However, properly interpreted tests can make CSF a key tool in the diagnosis of a variety of diseases. The present study aimed to etiologic diagnosis bacterial meningitis in a national paediatric hospital in Bangladesh using CSF culture method, and also to evaluate the usefulness of various laboratory procedures in diagnosing bacterial meningitis.
Bacterial meningitis remains a very important disease worldwide. From its original recognition in 1805 until the early 1900, bacterial meningitis was virtually 100% fatal (Quagliarello VJ et al., 1997). According to a World Health Organization estimate, approximately 171,000 people worldwide die from bacterial meningitis each year. Even with antimicrobial treatment, fatality rates are as high as 5-10% in the developed world. The incidence and mortality rates are much higher in third- world countries (World Health Organization, 2004.). Between 10% and 20% of those who do survive bacterial meningitis suffer permanent damage such as mental retardation, deafness, or epilepsy. Addition to the tragedy is the fact that these deaths could have been avoided; either through vaccination or by accurate diagnosis and rapid intervention (Babiker et al., 1984).
Acute bacterial meningitis is one of the most severe infectious diseases in the childhood. The global burden of the disease is high. Apart from epidemic, at least 1.2 million cases of meningitis are estimated to occur every year with 135,000 deaths (Tunkel et al., 1995). In developing countries like Bangladesh, it is a leading cause of bacterial meningitis (Salisbury, 1998), responsible for over 200,000 cases and more than 40,000 deaths annually (Salisbury, 1998 and Mulholland K, et al.1997). The disease is seen more in children than adults (Babiker et al., 1984). It is caused by a variety of organisms but the most important ones are Haemophilus influenzae, Neisseria meningitidis and Streptococcus pneumoniae in children and adults (Salisbury DM. and Dagbjartsson A et al., 1997). Other bacteria that have been less frequently implicated as pathogens include Streptococci; Gram-negative enteric or related organisms like Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa had been isolated infrequently from meningitis cases in Bangladesh (Hoque MM et al., 2006). The prevalence of these organisms varies from place to place, by age and season (Durand et al.1993). The specific pathogen causing bacterial meningitis varies around the world (Sigurdardottir et al., 1997, Tunkel et al., 2004 & Hussein et al., 2000). However, there is predominance of Gram- negative organisms as the etiological agents of bacterial meningitis (Van de Beek et al., 2004).
To reduce death or predominant neurological sequelae as much as possible, a fast and correct diagnosis is of the utmost importance. The current standard for the diagnosis of bacterial meningitis is microscopic examination and subsequent culture of cerebrospinal fluid (CSF) (Schuurman et al., 2004). Since meningitis is potentially hazardous disease of childhood, diagnostic tests are readily available, easy to interpret and simple to perform are of paramount importance. Lumber puncture is frequently performed in primary care (Wiswell et al., 1995; Shattuck et al., 1992 and Visser et al., 1980). Commonly performed laboratory tests on CSF include protein and glucose levels, cell count and differential, microscopic examination, and culture (Seehusen et al., 2003). However, properly interpreted tests can make CSF a key tool in the diagnosis of a variety of diseases. Culture is the gold standard for determining the causative organism in meningitis (Seehusen et al., 2003). The present study aimed to diagnosis bacterial meningitis using CSF culture method, to assess the prevalence and pattern of antimicrobial resistance of the isolated aetiological agents for proper selection of antibiotic therapy, and also to evaluate the usefulness of various laboratory procedures in diagnosing bacterial meningitis.
Bacterial meningitis is the most common and notable infection of the central nervous system, can progress rapidly, and can result in death or permanent debilitation. Not surprisingly, this infection justifiably elicits strong emotional responses and, hopefully, immediate medical intervention. The advent and widespread use of antibacterial agents in the treatment of meningitis have drastically reduced the mortality caused by this disease.
The majority of patients with bacterial meningitis survive, but neurological sequelae occur in as many as one-third of all survivors (especially newborns and children) (Saez-Llorens et al., 1990 & Saez-Llorens et al., 1991) Bacterial meningitis is much more common in developing countries than in the United States.
Meningitis is inflammation of the meninges that results in the occurrence of meningeal symptoms (e.g., headache, nuchal rigidity, and photophobia) and an increased number of white blood cells in the cerebrospinal fluid (CSF), i.e., pleocytosis (Razonable et al., 2005). Numerous infectious and non-infectious causes of meningitis are existed1. Acute bacterial meningitis denotes a bacterial cause of this syndrome. Haemophilus influenzae, Neisseria meningitidis and Streptococcus pneumoniae are the most common causes of bacterial meningitis in children (Schlech et al., 1985). The prevalence of these organisms varies from place to place, by age and by season (Schlech et al., 1985), but N. meningitidis is more often the commonest cause of meningeal infection (Bell et al., 1971) with S. pneumoniae (Spink et al., 1960 ) and H. influenzae (McGowan, 1974.) being second and third respectively. However, the order is reverse in a study in Bangladesh (Saha et al., 1997).
Children are most likely to get meningitis during their first year (Schlech et al., 1985; Health PT, et al. 2003 & Panjarathinam et al. 1993). Children who were infected as neonates had more health and developmental problems than those who had meningitis when they were older than one month (Bedford H, et al. 2001). The rates of severe or moderate disability differ widely between children infected with different organisms (Bedford et al. 2001). Infection with S. pneumoniae has been reported to be associated with a higher rate of disability than infection with H. influenzae and N. meningitides (Bedford et al., 2001; Baraff et al., 1993, McIntyre et al 1993 & Grimwood et al., 1995).
Bacterial meningitis has high mortality rate. Prior to the introduction of antibiotics in the 1940S, case fatality rates for epidemic and endemic bacterial meningitis exceeded 70% (Rao et al., 1998). Since then, antibiotic use has reduced case fatality rates for meningitis caused by most bacteria to 25% or less, but no further reduction has been documented in the past two or three decades (Panjarathinam et al., 1993 & Rao et al., 1998). Despite advances in vaccine development and chemoprophylaxis, bacterial meningitis remains a major cause of death and long term neurological disabilities, such as mental retardation, convulsions and hydrocephalus. These are best prevented by early diagnosis and appropriate treatment of the disease. Although lower mortality rates have been reported in industrialized countries such as the United States of America (2.6%) (Pomeroy et al., 1990), higher rates have been reported in some developing countries and countries in the Middle East, such as Turkey (38%) (Gurses N.1997), Saudi Arabia (14.7%) (Srair et al., 1992), Sudan (28.6%) (Ahmed et al., 1996) and India (21.8%) (Deivananyagam et al., 1993). Of the developing countries, the case fatality rate of 13.0% in the Libyan Arab Jamahiriya is not the highest among the world reports (Rao et al., 1998).
Although it is widely acknowledged that the consequences of meningitis in infancy may be severe, there are few studies that focus on the aetiologic diagnosis of bacterial meningitis (Saha et al., 1997, Rahman et al., 1990, Saha et al., 2003, Saha et al., 2005 and Hoque et al., 2006), and no reliable data from large prospective studies that focus on the outcome of infection in infancy or fatality in Bangladesh. Since meningitis is potentially hazardous disease of childhood, diagnostic tests that are readily available, easy to interpret and simple to perform are of paramount importance. Lumbar puncture is frequently performed in primary care (Wiswell et al., 1995, Visser et al., 1980 and Shattuck et al., 1992). Commonly performed tests on cerebrospinal fluid (CSF) include protein and glucose levels, cell counts and differential, microscopic examination, and culture (Razonable et al., 2005 & Seehusen et al., 2003). Culture is the “gold standard” for determining the causative organism in meningitis (Seehusen et al., 2003). However, properly interpreted tests can make CSF a key tool in the diagnosis of a variety of diseases. The present study aimed to identify the aetiologic agents of bacterial meningitis in a pediatric hospital in Bangladesh using CSF Culture method, and also to evaluate the usefulness various laboratory procedures in diagnosing bacterial meningitis.
Acute bacterial meningitis is an infection of the nervous system that results in inflammation of the meninges, the membranes that surround the brain and spinal cord. It occurs in the US with an annual incidence of approximately three cases per 100,000 persons (Attia et al., 1999). Overall mortality attributable to bacterial meningitis in various case series has ranged from 15 % to 21 % (Sigurdardottir et al., 1997). Hence, it is extremely important that clinicians be knowledgeable of this condition for a diagnosis to be made and for timely therapy to be instituted.
Bacterial meningitis is a serious disease with high morbidity and mortality. To reduce death or permanent neurological sequelae as much as possible, a fast and correct diagnosis is of the most importance. The current standard for the diagnosis of bacterial meningitis is microscopic examination and subsequent culture of CSF. However, this approach might have some disadvantages with regard to the desired rapidity and sensitivity. Results of culture may only be available after 24 to 48 h and sometimes for instance, when the number of viable organisms in the CSF is low it may take even longer. Moreover the sensitivity of microscopic examination and culture of CSF can be debated.
First, bacterial concentration in the CSF has a profound effect on the results of microscopy. Regardless of the type of organisms in the CSF, the percentage of positive microscopic results is only 25% with <103 cfu/ml and 60% in the range of 103 to 105 cfu/ml.
Second, in an extensive study over a period of 27 years, it appeared that culture might miss the diagnosis of bacterial meningitis in at least 13% of cases. Acknowledged reasons for this lack in the sensitivity are CSF obtained after the start of antibiotic treatment and meningitis due to the fastidious or slow growing microorganisms (Schuurman et al., 1998)
Bacterial meningitis continues to be one of the most serious infectious diseases experienced during childhood and despite the availability of newer antibiotics and a greater understanding of the pathogenesis of the disease, a considerable mortality and morbidity may still be experienced. Knowledge of the causative organisms and their antibiotics sensitivity in a region may be of importance.
First, when considering empirical therapy when a causative organism cannot be identified. Secondly, in view of the increasing availability of vaccines offering protection against this potentially devastating disease (Donald et al., 1996)
Bacterial meningitis is relatively common, can progress rapidly, and can result in death or permanent debilitation. This infection justifiably elicits strong emotional reactions and, hopefully, immediate medical intervention. This review is a brief presentation of the pathogenesis of bacterial meningitis and a review of current knowledge, literature, and recommendations on the subject of laboratory diagnosis of bacterial meningitis. Those who work in clinical microbiology laboratories should be familiar with the tests used in detecting bacteria and bacterial antigens in cerebrospinal fluid (CSF) and should always have the utmost appreciation for the fact that results of such tests must always be reported immediately. Academic and practical aspects of the laboratory diagnosis of bacterial meningitis presented in this review include the following: anatomy of the meninges; pathogenesis; changes in the composition of CSF; etiological agents; processing CSF; microscopic examination of CSF; culturing CSF; methods of detecting bacterial antigens and bacterial components in CSF (counter-immunoelectrophoresis, coagglutination, latex agglutination, enzyme-linked immunosorbent assay, Limulus amebocyte lysate assay, and gas-liquid chromatography); use of the polymerase chain reaction; and practical considerations for testing CSF for bacterial antigens (Gray et al.,1992)
In acute bacterial meningitis, the classic symptoms and signs of meningeal irritation are common, but these signs may be present in other diseases like acute viral meningitis, tuberculous meningitis, subarachnoid hemorrhage, etc. Most of the patients of acute meningitis usually receive broad spectrum antimicrobial therapy before any diagnostic approach taken. The CSF should be examined in every patient in whom the clinical findings are consistent with the possibility of meningitis. Alternative methods of CSF study have been developed which may be useful in patients commenced with antibiotic therapy before lumber puncture.
Where culture is usually negative, detection of soluble bacterial antigens can help to reach a diagnosis. The Latex Particle Agglutination Test (LPAT) has been introduced for this purpose because it can detect comparatively very small quantity of antigens present.
Although specificity of these tests is good, sensitivity is not better than a Gram stain. Therefore, negative results for a specific bacterial antigen do not rule out bacterial meningitis (Phillips et al., 1991).
The main limitation of LPAT is that it is positive only in the presence of specific polysaccharide surface antigens for H.influenzae type b (Hib), S. pneumoniae, E. coli, group B Streptococcus and N. meningitidis A, C, Y, W-135 antigens, while any other bacteria remain undetected. The Gram’s staining method does not have this limitation. To optimize cost benefit ratio, the test should not be indiscriminately used. Thus, the LAPT should be done routinely when positive Gram stain and abnormal CSF values (WBC counts, glucose, protein concentrations and other markers) indicated bacterial meningitis.
The antigens of common meningeal pathogens e.g. H. influenzae type b (Hib), S. pneumoniae, E. coli, group B Streptococcus and N. meningitidis are detected by the LPAT. The diagnosis of bacterial meningitis caused by specific pathogens is established by positive LPAT, but a negative test does not rule out other bacterial meningitis cases. A broad-spectrum antibiotic coverage is usually recommended as an initial treatment for suspected bacterial meningitis, which is very costly. The LPAT can diagnose these specific bacterial pathogens and specific antibiotic therapy can be given to reduce the emergence of bacterial resistance (Begum N, et al., 2007).
Bacterial meningitis in infants and children is a serious clinical entity with signs and symptoms that commonly do not allow distinguishing the diagnosis and the causative agents. Acute meningitis is a common infection, predominantly aseptic (82– 90%), but when of bacterial origin (10-20%), it is infrequently associated with severe neurologically sequelae, especially when the diagnosis and treatment are late (Nigrovic et al., 2002 & Tatara et al., 2000). As it is difficult to distinguish between bacterial and aseptic meningitis in the initial state, most authors have recommended rapid initiation of antibiotics in children with acute meningitis, with conventional therapy until cerebrospinal(CSF)culture results become available, 48-72 hours later (Saez-Uorens et al., 2003 & El Bashir et al., 2003).The pattern of bacterial meningitis and its treatment during neonatal periods may over lap, especially in the first one to three months old in whom group B streptococcus, Haemophilus influenza-type b , meningococcus and pneumococcus may all produce meningitis (Tunkel et al., 2004 & Feigin et al., 1992). In children more than 3 months of age H. influenzae, Streptococcus pneumoniae, Neiseria meningitiditis are the commonest causative organism of bacterial meningitis. The aim of this study is to study the microbiological profile of CSF in childhood meningitis, over a period of one year (Saez-Uorens et al., 1990).
In healthy children, the three most common organisms causing acute bacterial meningitis are S.pneumoniae, N meningitidis, and H. influenzae type b (Hib) (Freedom et al., 2001). Although Hib is the commonest causative agent), with the availability of Hib conjugate vaccine, the current likely hood of Hib meningitis in a child who has received at least two doses of vaccine was extremely rare. Lumbar puncture is the gold standard for the diagnosis and should be done in all suspected cases of meningitis unless contraindicated (Freedom et al., 2001). It helps to distinguish the microbial etiology of meningitis and encephalitis, and to rule out non-infectious causes of disease (Kneen et al., 2002). The myth about lumbar puncture complications among parents has to be resolved by the physician in order to get the consent to do the procedure
Development of bacterial meningitis progress through the following steps:
Bacterial colonization of the nasopharynx
Mucosal inflammation and penetration into the blood stain
Intravascular multiplication and entrance through the blood brain barer.
Generation of inflammation within the subarachnoid space
Neuronal cell injury and auditory nerve damage.
Children with bacterial meningitis present in one of the following pattern:
The most common and insidious form with non specific symptoms that progress over 2 to 5 days before meningitis is diagnosed.
A more common rapid form, in which symptoms and signs of meningitis progress over one or two days.
A fulminant course, with rapid deterioration and shock early in the course of illness.
On physical examination, the fontanel of an infant may be bulging, presumably indicating increased intra cranial pressure; this sign is neither highly sensitive nor specific for meningitis but always requires evaluation. Most specific physical findings of meningitis are Kernig’s and Brundenzki sign and neck stiffness. Papilledema is uncommon in a child with a uncomplicated meningitis and if present, suggest another cause such as subdural effusion, brain abscesses etc (Saez-Uorens et al., 1990).
Petechial or purpuric rash and shock are classically associated with meningococcal meningitis but also can be occasionally caused by H. influenzae or S. pneumoniae (Oastenbrink et al., 2004 & Bingen et al., 2005).
Table 1: Laboratory values of components of CSF from healthy persons and from patients with meningitisa
| Traits | CSF laboratory value | |||
| Protein Glucose Leukocytes Predominant cell
(mg/dl) (mg/dl)b (perml ) |
||||
| Healthy persons | ||||
| Newborns | 15 – 170 | 34 – 119 | 0 – 30 | |
| Adults | 15 – 50 | 40 – 80 | 0 – 10 | Lymphocytes (63-99)
Monocytes (3-37) PMN (0-15) |
| Adult patients with:
Bacterial meningitis Fungal meningitis Viral or aseptic meningitis |
100
Increased <100 |
40
<30 Normalc |
1,000
Increased <500 |
PMN(>50)
Lymphocytes PMN(early) and lymphocytes (late) |
a. Data are commonly observed values. Notable exceptions to these values and overlap of values elicited by different etiological agents are not uncommon. PMN, polymorphonuclear leukocytes.
b. The CSF glucose/serum glucose ratio usually is 0.6 (adults) or 0.74 to 0.96 (neonates and preterm babies). In-patients with bacterial meningitis, the ratios usually are <0.5 (adults) and <0.6 (neonates and preterm babies).
c. Lower than normal glucose concentrations have been observed during some noninfectious disease processes and in some patients with viral meningoencephalitis due to herpesviruses, varicella-zoster virus, mumps virus, lymphocytic choriome
1.4 ETIOLOGICAL AGENTS OF BACTERIAL MENINGITIS
The results of national surveillance studies have shown that both the etiological agents and mortality rates (0 to 54%) of bacterial meningitis depend on the season of the year and the age, sex, ethnic background, and geographic location of the patient (McGee et al., 1990, Schlech et al., 1985 and Wenger et al., 1990). multistate surveillance study of the etiological agents of bacterial meningitis (Wenger et al., 1990). H. influenzae was the most frequent cause of bacterial meningitis (2.9 cases per 100,000 populations) and, paradoxically, was associated with the lowest fatality rate (3%) of the five most frequent bacterial agents.
Table 3: Shows the results of a 1986a
| Bacterium | No. (%)
of cases |
Incidence
(cases/100,000) |
Case fatality
Rate (%) |
| Haemophilus influenzae | 964(45) | 2.9 | 3 |
| Streptococcus pneumoniae | 379(18) | 1.1 | 19 |
| Neisseria meningitidis | 293(14) | 0.9 | 13 |
| Streptococcus group B | 122(5) | 0.4 | 12 |
| Listeria monocytogenes | 69(3) | 0.2 | 22 |
| Otherb | 331(15) | 1.0 | 18 |
aData were obtained from a surveillance study by Wenger et al.,1990 and are used with permission of the publisher.
bOther bacteria include Streptococcus spp. other than group B, S. aureus, E. coli, S. epidermidis, Klebsiella spp., Enterobacter spp., Serratia spp., and Acinetobacter spp.
On the other hand, Listeria monocytogenes was reported relatively infrequently (0.2 case per 100,000 population) but had the highest fatality rate (22%). Table 4 contains additional data from the aforementioned 1986 study and shows the distribution of etiological agents of bacterial meningitis in five commonly defined age groups. Streptococcus group B (Streptococcus agalactiae), H. influenzae, N. meningitidis, and Streptococcus pneumoniae were the leading causes of bacterial meningitis in neonates, young children, young adults, and adults and senior adults, respectively. Certain elements of a patient’s history (e.g. predisposing factors, medical condition, epidemiology, occupation, and immune status) can suggest specific bacterial agents of meningitis (Isenberg et al., 1991; Kaufman et al., 1990 and McGee et al., 1990). Unusual and rare bacteria that have been reported to cause meningitis include Bacteroides fragilis (Odugbemi et al., 1985).
TABLE 4: Etiological agents of bacterial meningitis in five age groups (1986)a
| % of casesb caused by: | ||||||
| Age | Streptococ-cus groupB | L.
monocytogenes |
H.
influenzae |
S.
pneumoniae |
N.
meningitidis |
Otherc |
| 0-1 mo | 49 | 9 | 5 | 3 | 1 | 33 |
| 2mo-4 yr | 2 | 70 | 10 | 13 | 5 | |
| 5-29 yr | 2 | 2 | 8 | 17 | 42 | 29 |
| 30-59 yr | 4 | 6 | 5 | 37 | 10 | 38 |
| ?60 yr | 3 | 14 | 4 | 48 | 3 | 28 |
aData were obtained from a surveillance study by Wenger et al., 1990 and are used with permission of the publisher.
bThe percentages were extrapolated by us from the data in reference (Wenger et al., 1990 ).
cOther bacteria include Streptococcus spp. other than group B, S. aureus,
E. coli, S. epidermidis, Klebsiella spp., Enterobacter spp., Serratia spp., and Acinetobacter spp
1.5 STREPTOCOCCUS PNEUMONIAE
1.5.1 Introduction
Streptococcus pneumoniae is one of the leading causes of morbidity and mortality throughout the world including Bangladesh. It is most common cause of community acquired bacterial pneumonia and the second most common cause of bacterial meningitis in children less than five years of age in Bangladesh and across the developing world. It also causes abdominal infection, bactermia, otitis media and various diseases. Most children experience some form of pneumococcal infections and some develops sepsis or meningitis. Despite over a century of research, many aspects of pneumococcal disease remain obscure. The continued frequency and severity of pneumococcal disease, coupled with the knowledge that use of antimicrobial agents does not invariably present or death and the recent emergence of strains of S. pneumoniae resistant to most antimicrobial agents, all severe to underscore the need for better understanding of pneumococcal infections. Particular attention is presently concentrated on efforts to prevent these in infections by development of appropriate vaccine (Feigin and Cherry et al., 2000).
Meningitis occupies only a small portion of the spectrum of diseases caused by pneumococcus. The organism has positioned itself as the leading cause of mortality inunder-five children in developing countries, mainly by causing pneumonia, a silent killer of third world children.
World Health Organization (WHO) calculates that the organism, as a whole, by causing pneumonia, meningitis, sepsis and other diseases, kills more than 800,000 under-five children worldwide (WHO. Pneumococcal vaccines.Wkly Epidemiol Record 2003, Williams,et al., Lancet ID2002) and 90% of them are in the developing part of the world (httt://www.preventpneumo.org/disease_vaccines).
Approximately 20% to 40% of global total of four million deaths of children bellow five years of age occurred from pneumonia globally per year (Saha et al., 1999). Such a fetal causing agent (pneumonia) of human morbidity and mortality required special study and research in locally and globally but not a perfect study has been done in Bangladesh in this field. By considering the situation our study on pneumococci has special importance. Various antibiotic (penicillin) resistance or decreased susceptibility to antibiotics among the clinical isolates of S. pneumoniae became a threatening problem, which increase, dramatically in the last decade (Felminham et al., 1999). Bangladesh is not devoid of this situation due to the worldwide migration of the antibiotic resistant clones of S. pneumoniae along with the migration of the pneumococcal carrier or pneumococcal infected individuals. How fast the antibiotic resistant pneumococcus has been spread and grown up, could be understand by the following examples.
In England and Wales, for example, erythromycin resistant rate increased from 2.8% to 8.6% in the year between 1990 to 1995 (Goldsmith et al., 1997). In a study in central Italy, erythromycin resistant rate increased from 7.1% to 32.8% in the year between 1993 to 1997 (Oster et al. 1999). In France, resistance to erythromycin was first described in 1976 and increased to 20% in 1984 then to 29% in 1990 (Geslin et al., 1992). On the other hand, the first case of clinically significant penicillin resistant isolates was reported in Australia in 1967 (Hansman et al., 1967) an abrupt increase of penicillin resistance among pneumococcal was reported in last decade. For instance, the frequency of penicillin resistance in Italy increases from 5.5% to 7.7% in the year between 1993 to 1996 (Marchese et al., 1998). The preliminary report from Bangladesh showed that 10% of S. pneumoniae strains were resistant to penicillin (Saha et al., 1992). If the above rate of antibiotic resistance is continued, all the antibiotics designed for S. pneumoniae will be infective against pneumococcal infection and that would be a threatening condition for the human health. But studies on the prevalence, serotypes and detection of antibiotics resistant clones and their distribution are few in numbers in Bangladesh. To understand and contain this crucial problem first we have to know the prevalent serotypes, patterns of antibiotics resistance and the clonal distribution of pneumococcal clinical isolates from the individuals in Bangladesh.
Prospective neurodevelopmental assessments of pneumococcal meningitis patients in Bangladesh, 3-4 months and 12-24 months after discharge from the hospital, revealed that about 65% and 49% of cases survived with one or more impairments and permanent disabilities respectively, including deafness, vision loss, mental shortfall, and psychomotor deficits. Other studies in India (Gupta V, 1993), Pakistan (Qazi SA, et al., 1997), Sudan (Salih MA, et al., 1991), and Vanuatu (Carrol KJ, et al., 1994) have shown similar results.
This high prevalence of disability from pyogenic meningitis cases can be attributedto poor care-seeking behavior, and delay in diagnosis and treatment of meningitis (Salih MA, et al., 1991 and Richardson MP, et al., 1997).
1.5.2 Bacteriology
Streptococcus pneumoniae along with many other species of Streptococci, e.g., S. pyogenes; S. agalactiae, etc. belongs to the pyogenic category of Streptococcus genus. Table 1.6 shows the comparison between the pyogenic Streptococcal species (Bergy’s manual of systemic bacteriology 1998).
1.5.3 Morphology
Streptococcus pneumoniae is encapsulated Gram-positive diplococci, oval or spherical in shape, and 0.5 to 1.25 mm in diameter. It occurs singly in pairs and short chains occasionally as individual cocci (Willett et al., 1992). Continued laboratory cultivation, especially on unfavorable media or in the presence of type specific antibody, leads to the formation of larger chains or may be found in short chains and particularly with a low Mg++ concentration. Gram-positive reaction of young cells may be lost as culture ages and subsequently strains Gram-negative.
1.5.4 Biochemical properties
Surface-active agents, such as bile salts or sodium deoxy-cholate stimulate autolysis. The test for “bile solubility” is useful in identification of pneumococci. They are inulin fermenters. Deoxy-cholate activates an amidase that splits the tetra peptide from muramic acid in peptidoglycan. This reaction is properly regulated; normally function in wall morphogenesis and cell division.
1.5.5 Cultural characteristics
The Streptococcus pneumoniae has complex nutritional requirements. It can be grown on chemically defined synthetic media but for primary isolation and routine culture, enriched infusion agar and broth such as trypticase soy brain heart infusion enriched with 5% defibrinated sheep or goat or horse blood, is recommended (Willett, 1992). The optimum PH and temperature for growth is 7.4 to7.8 and 370C, respectively. All pneumococcal strains require an increased CO2, concentration for primary isolation on solid agar media. A candle extinction jar or CO2 incubator should be used for this purpose. Colonies on blood agar plates are small, smooth and transparent. Low convex while tiny, they become flattened or depressed centrally, showing the “draughtsman form.” As they grow older. Some strain e,g. of type 3, which form large capsule, tent to remain convex (Mackie & Mc. Cartney 1996). Uncapsulated strains produce small rough colony. A partial clearance of blood and a greenish discoloration (?-hemolysis) is produced underneath. A narrow zone around the colonies formed when they are incubated aerobically. Aerobic incubation results in ?-hemolysis due to pneumolysin ‘O’ activity. Unlike other Streptococcus, S. pneumoniae requires cholone for growth in defined media. Ethanolamine replaces choline but not on a molar basis. Ethanolamine substitution during growth leads to a number of physiological defects including:
Resistance to autolysis,
Aberrant cell division,
Incompetence in transformation, and
Phage resistance.
1.5.6 Physiology
Reducing agents are essential for S. pneumoniae. Most strains require at least 4 of the B vitamins for growth as well as adenine, guanine, and uracil and 7 to 10 amino acids. S. pneumoniae is a facultative aerobic and its energy-yielding metabolism is fermentative, yielding primary low level of lactic acid. Under aerobic condition a significant amount of hydrogen peroxide (H2O2) accumulates, with some degree of acetic and formic acids. S. pneumoniae does not produce catalase or peroxidase and the accumulation of hydrogen peroxide kill the organism unless catalase is provided by the addition of RBC (Red blood cell) to the culture media (Willett, 1992). The organism tents to die fairly quickly in cultures e.g. in course of a day or two, particularly in aerobic cultures media without blood. The dead organism tents to undergo autolysis (Mackie et al., 1996).
1.5.7 Diagnosis of pneumococcal infections
Demonstrating the presence of pneumococcal in a specimen of sputum, lung aspirate, CSF, urine or blood directly by Gram-stain and culture, and then identifying the culture in an optochin sensitivity test generally do diagnosis of S. pneumoniae. The approach is likely to be successful when a heavily infected specimen is collected early in the ilness and before the start of antibiotic therapy. Now a day, because of the emerging problem of antibiotic resistant pneumococci, definitive diagnosis is absolutely necessary. There are four ways of detection of pneumococci:
Microbiological identification
Serological identification
Molecular biological test
Animal pathogenecity test
1.6 MICROBIOLOGICAL IDENTIFICATION
1.6.1 Gram-Stain
Under microscopic examination, pneumococci appear as Gram-positive diplococci (Bergy’s manual of systemic bacteriology 1998, vol.II).
1.6.2 Optochin sensitivity test
Commercially available optochin disks are applied to a quarter of a blood agar plate that has been streaked with a few colonies of the organism to be tested. After overnight incubation at 350C in either candle-extinction jar or a CO2 incubator, inhibition zones are measured. Zone of >14 mm with a 6 mm disc or >16mm with an mm disk indicates inhibition and identify the isolates as S. pneumoniae. Isolates laying smaller zone of inhibition should be subjected to an additional test for bile solubility to confirm their identities (Willett et al., 1992)
1.6.3 Bile solubility test
Pneumococci are soluble in bile but viridans and other Streptococci are not (Mackie and Mc. Cartney, 1996). This test is based on the presence of an autolytic amidase in pneumococci, which cleaves the bond between alanine and muramic acid in the peptidoglycan. Surface-active agents such as bile or bile salt, resulting in lysis of the organism, activate this amidase. For testing a neutral pH, 10% deoxycholate and viable young organisms are required (Willett et al., 1992).
1.6.4 Quellung Reaction
This is the most useful and rapid method for identification of S. pneumoniae. The test not only identifies an organism as a S.pneumoniae but also specifies its type (Willett et al., 1992). The Quellung or “capsular swelling” reaction is actually a reaction between the bacterial capsular polysaccharide and its homologous antiserum.
1.7 SEROLOGICAL IDENTIFICATION
1.7.1 Counter current immunoelectrophoresis (CIE)
The method described by Dulake (1979), is an especially sensitive method for detecting capsular antigen and may give a positive result when the coagglutination test is negative (Mackie & Mc. Cartney, 1996). The methodology of CIE is based on the fact that most soluble bacterial antigens are negatively charged in slightly alkaline media (pH 8.2- 8.6). Under the same condition antibody (usually rabbit Ig) is neutralized or slightly changed. When an electric field is applied the resulting antigen-antibody complex forms a precipitation line, in between two walls. By varying antibody, the bacterial antigen can be determined.
1.8 MOLECULAR BIOLOGICAL METHOD
1.8.1 Polymerase chain reaction (PCR)
The polymerase chain reaction (PCR) is a technique that can be used to find very low quantities of an infectious agent present in clinical sample by increasing the quantity of a specific nucleotide sequence continued within the organism by a process of detecting in-vitro DNA synthesis. With the advent of PCR technique (Mullis & Falona, 1987) several workers attempted to utilize this technique to detect S. pneumoniae from clinical samples. The pioneer in these cases is Rudolph et al., (1993). They developed a nested PCR protocol, and used blood sample from patient’s culture proven bacteremia. The next work for the detection of S. pneumoniae DNA in blood cultures by PCR was done by Hassan-King et al., 1994. The objective of his study was to develop a PCR assay that would improve the frequency of detection of S. pneumoniae in patients with septicemia. In August 1994 Zhang et al published his work on detection of S. pneumoniae penicillin binding protein 2B gene. In this study QIA Amp Kit was used for the processing of whole blood. Salo et al. From Finland in 1994 developed a PCR diagnosis method of S. pneumoniae by amplification of pneumolysin gene fragment in serum. They used a nested PCR strategy for the detection assay. Their study identified, besides all culture positive samples, 6 culture-negative samples for S. pneumoniae in 100 healthy patients by PCR. In February 1996, Hassan-King et al. published a multiplex PCR assay for the simultaneous detection of S. pneumoniae and H. influenzae type b. They used the primers from the autolysin’s gene and used blood as sample. A semi-nested PCR strategy for the detection of penicillin resistant S. pneumoniae from CSF was developed by Mignon et al. (1997) from South Africa. Very recently, an article from Israel has shown a prospective study for the detection of pneumococcal DNA in sera of children by single PCR (Dagon et al., 1998).
1.8.2 Identification by DNA probes hybridization
Nucleic acid hybridization tests often reduce the time necessary to identify microorganism. They also allow laboratories to increase the number of types of pathogens that can be easily detected and identified. Nucleic acid probes are segments of DNA or RNA that have been labeled with enzymes, or radioisotopes and can bind with high specificity to complementary sequences of nucleic acid. Oligonucleotide probes can be chemically synthesized and purified with relative ease. DNA probe from lyt A gene was constructed and is used for S. pneumoniae detection. Dot blot hybridization is used for the assay. For the process 0.5N NaOH denatured the probe for lyt a gene. Denatured DNA (0.1 ml) was applied to 96 wells Bio-Dot apparatus containing the sample (Pozzi et al., 1989).
1.8.3 Drug resistance in Streptococcus pneumoniae
Following the introduction of penicillin in 1940, pneumococci wre regarded as uniformly sensitive to this antibiotic. This brief was so well establish that the use of in vitro susceptibility testing was not considered necessary (Zighelboin et al., 1981, Hussein et al., 1989). This idea persisted until 1967 when the first strain showing increased resistance to penicillin was isolated (Hansman and Bullen, 1967).
Various patterns of S. pneumoniae resistant to drug other than penicillin had been reported. Strains resistant to penicillin, tetracycline, erythromycin, and chloramphenical in various combinations have been reported from different parts of the world (Hansman et al, 1974 & Cates et al, 1978). In the present study, resistance to tetracycline, Co-trimoxazole, chloramphenical and erythromycin was 70%, 43%, 12% and 4% respectively. Tetracycline resistant pneumococci strains averaged 13% in 1975 in England (Repoted of an Adhoe study Group on antibiotic resistance, 1977), 58% in 1983 in Hong Kong (Long et al., 1983), 67% in spain in 1982 (Casal, 1982), 67% in 1987 in Saudi Arabia (Mahgoub and Hussein, 1987) and 70% in the present study. Most studies indicate that approximately 10% of pneumococci are resistant to co-trimoxazole (Malatovic et al., 1981, Michel et al., 1983 and Henderson et al., 1988).
In Bangladesh, the pneumococcus has been identified as the predominant cause of meningitis and pneumonia in children (Khan et al., 1989, Saha et al., 1988) and there are no reports of penicillin resistance of this organism. In a study all strains were sensitive to penicillin (Saha et al., 1988). And in other (Khan et al., 1989) no sensitivity pattern was shown in the first study, however, the total number of strains was only eight and disc diffusion method was used, with penicillin disc (10 micro gram) to detect resistance to the drug. The validity of this test is questionable since it does not detect penicillin resistant pneumococcus strains. Since 1989 Dr. S.K. Saha and his colleagues screened 51strains of pneumococci isolated from cerebrospinal fluids (CSF) (39) and blood (12) for resistance to penicillin. The minimum inhibitory concentrations (MIC) were measured on Muller-Hinton agar containing 5% sheep blood and various concentrations of antibiotic (Saha et al., 1998). Of these 51 strains 2 (3.9%) were fully resistant (MIC>1.0 micro gram/ml) 4 (7.8&) were moderately resistant (MIC> 0.12-1.0 micro gram/ml). The remainder (88%) were sensitive to penicillin (MIC<0.06 micro gram/ml) (Saha et al., 1991). However, drug resistant pattern of pneumococci other than penicillin is not yet known in Bangladesh.
1.9 Prevention by vaccine
Despite, effective antimicrobial agents severe pneumococcal infections are common and an excessive number of deaths still occur. As a result, there is an urgent need for vaccines that reduce the mortality associated with infections caused by S. pneumoniae (Frank Shann 1995).
Polysaccharide vaccine
A polyvalent polysaccharide vaccine was developed for the prevention of serious pneumococcal infections.
Its wide spread use in adults “at risk” for pneumonia, immunocompromised with chronic diseases or over 65 years age can be expected to reduce mortality, morbidity and associated health care costs (Carol Brignoli Gable et al., 1990). The currently available pneumococcal vaccine contains purified capsular polysaccharide antigens of 23 serotypes of S. pneumoniae. Each 0.5 ml dose of vaccine contains 25 microorganisms of each polysaccharide antigen, compared with 50 micrograms of each in the 14-valent vaccines, which was replaced in 1983 (Mclyntyre et al., 1997).
Efficacy of the vaccine may decrease with increasing age time since vaccination. Among older adults, antibody levels may decrease after 6 years to levels that are not protective (Saha et al., 1996). Almost no response is seen in individuals with leukemia lymphoma or Hodgkin’s disease. Even infants below one year of age will respond to few serotypes but the antibody does not persist (Mclyntyre et al., 1997). A maternal immunization study by Staid et al., has shown that maternal antibody is passed on to the fetus in concentrations two to three fold higher than those of control infants. The burden of pneumococcal disease in developing countries is greater in children than 6 months os age, and therefore because these children are unlike to be fully immunized with pneumococcal conjugate vaccine before 4 months of age and the route of maternal immunization is one that deserves greatest attention (Fedman & Klugman, 1997).
1.10 Conjugate pneumococal vaccine
Coupling of pneumococcal polysaccharides to protein has been shown to enhance the immune system response to the polysaccharide moiety in animals, therapy resulting in protective immunity against S. pneumoniae. The immunological basis for the increased immunogenicity of polysaccharide-protein conjugates is related to the T-cell dependent character of these conjugates. Upon repeated immunization increased number of activated protein-specific thymic cells is thought to provide help to polysaccharide-specific B cells, resulting in their differentiation towards memory or plasma cells. Pneumolysin is to date the best studied pneumococcal protein. Virtually all clinical isolates of S. pneumoniae produce pneumolysin. Its primary structure is remarkable as well as being independent of capsular serotypes, geographic area, and time of isolation. Although some native pneumolysin has strong toxic effects several derivatives, which are nontoxic but retain immunogenicity and protective activity of the native protein has been engineered. These constructs therefore seem to meet many criteria for inclusion in a pneumococcal conjugate vaccine. The conjugate induced substantially higher antibody responses than did the polysacchiride alone. This strongly suggested that the carrier protein (Velasco et al., 1995) activate thymic cells.
1.11 Pneumococcal infection in Bangladesh
S. pneumoniae is the most prevalent cause of community acquired bacterial pneumonia, and of other respiratory infections throughout the world and in Bangladesh, being a developing country, is not an exception. Although not much work has beendone on this important pathogen in Bangladesh, the limited data suggested that it is commonest cause of child mortality and morbidity due to acute respiratory infections. One hundred sixty-five invasive S. pneumoniae strains were isolated from children under five years of age at Dhaka Shishu (children) Hospital during the period 1992 to 1995. Ninety-four (57%) of the strains were isolated from 412 pyogenic cerebrospinal fluid (CSF), an