Laboratory Diagnosis of Bacterial Diseases: 10 Main Types

This article throws light upon the ten main types of laboratory diagnosis of bacterial diseases. The types are: 1. Diphtheria 2. Enteric Fever 3. Isolation of the Organism 4. Bacillary Dysentery 5. Diarrhoeal Diseases 6. Bacterial Food Poisoning 7. Cholera 8. Urinary Tract Infections 9. Meningitis 10. Tuberculosis.

Bacterial Disease: Type # 1. Diphtheria:

The genus corynebacterium comprises of:

1. Toxin-producing: C. diphtheriae, C. ulcerans.

2. Non-pathogenic (commensals): C. hofmannii, C. xerosis. These are also called diphtheroids.

A. Bacteriological Studies:

For control measures and epidemiological studies laboratory confirmation of diphtheria is necessary. Specific treatment against diphtheria (antitoxin and antibiotic) should be given immediately after clinical diagnosis without waiting for laboratory findings. Two swabs are taken from the site of lesion (throat, nose or larynx): one for smear examination and other for culture.

1. Direct smear examination:

Smears stained by Albert’s stain show beaded slender rods in typical Chinese-letter pattern. The results of microscopic examination of clinical material may not always be reliable. Interpretation of the smear requires technical expertise. Hence smear examination alone is not sufficient for the diagnosis. Specimen should always be cultured.

2. Culture:

Swab is inoculated on the following culture media:

(a) Loeffler’s serum slope:

Growth appears in 6-8 hours but, if negative, the media will have to be incubated for 24 hours. The microscopic morpho­logy is best seen on Loeffler’s medium. Smear from culture stained with Albert stain show typical bacilli.

(b) Tellurite blood agar medium and ordinary blood agar medium:

It is incubated for 36-48 hours. Studies on colonies on tellurite medium is helpful in distin­guishing the biotypes of C. diphtheriae (Table 12.1). The colonies show characteristic gray-to-black colour on tellurite agar.

B. Virulence Test:

All isolates of C. diphtheriae should be tested for virulence for toxigenicity which can be done either in vivo (animal inoculation) or in vitro (Elek’s test).

1. Animal inoculation test:

Guinea pigs and rabbits are susceptible but rat and mice are resistant to the action of toxin.

(a) Subcutaneous test:

Overnight growth of Loeffler’s slope is first mixed with 2 to 5 ml nutrient broth and 0.3 ml from this emulsified growth is injected subcutaneously in thigh in two guinea pigs, one of which has received an intramuscular injection of 500 units of diphtheria antitoxin 18 to 24 hours before the test. The unprotected animal will die within 2 to 3 days.

At autopsy, other important findings include: gelatinous, haemorrhagic oedema and often with necrosis at the site of injection; draining lymph nodes-swollen; clear, cloudy or blood-stained pleural and peritoneal fluid; and abdominal viscera-congested. The adrenal gland finding (haemorrhage) is pathognomonic feature. This method is not usually followed because of wastage of animals.

(b) Intra-cutaneous test:

Two guinea pigs or rabbits are injected intracutaneously with 0.2 ml emulsion from growth in Loeffler’s serum medium, one of which is protected with 500 units of antitoxin the previous day (control animal) and to the other animal 50 units of diphtheria antitoxin is injected intraperitoneally simultaneously with the skin test in order to prevent abrupt death of the animal.

The unprotected animal develops local erythematous lesion at the site of injection in 48-72 hours but there is no change in the control animal. By intra-cutaneous method, 8 to 10 cultures can be tested on a pair of animals and the animals do not die.

2. In vitro test (Elek’s Agar-gel precipitation test):

This in vitro immune-diffusion test, described by Elek (1949), provides a rapid and economic alternative to in vivo test. A rectangular strip of filter paper soaked in antitoxin (1,000 units/ml) is placed on the surface of a serum agar plate containing 20% horse serum while the medium is still in fluid form.

When the medium solidifies, the testing strain is streaked across the plate at right angles to the filter paper strip and then incubated at 37°C for 24 to 48 hours. Toxin produced by bacterial growth diffuses in agar medium and produces line of precipitation where it meets antitoxin molecules (dispersed from the filter paper) in optimum concentration (Fig. 12.1).

Usually the result can be read within 48 hours, sometimes precipitin lines may take up to 72 hours to develop. This technique is not only very convenient but also economical. The lines of pre­cipitate radiate from the intersection of the strip and the bacterial growth.

3. Tissue culture test:

This is done by incorpora­tion of test bacteria into an agar overlay of cell culture monolayers The toxin produced diffuses into the underlying cells causing death of the cells.

The important distinguishing features of C diph­theriae, Table 3.1 .

Bacterial Disease: Type # 2. Enteric Fever:

Salmonella typhi causes typhoid fever and S. paratyphi A, B and C cause paratyphoid fever. Enteric fever includes both typhoid and para­typhoid fever. Other important pathogenic salmonellae include enterocolitis group (caused by S. typhimurium, S. enteritidis, S. dublin etc.) and septi­caemia group (caused by S. enteritidis).

Culture and isolation of salmonellae are essential to confirm a diagnosis. Identification of the isolate is based on biochemical characteristics and serology. The optimal specimen for culture varies with presen­tation.

(a) In a classic case of salmonella gastroenteritis a faecal sample is best. Patient’s vomitus and suspe­cted foodstuffs are also useful.

(b) In a suspected case of enteric fever, blood culture or bone marrow aspirate is most likely to yield positive results. Later, with intestinal and renal involvement, culture of faeces and urine is indicated.

General blood picture in enteric fever may show leucopenia (leucocyte count, 2,000-2,500 cumm) with relative lymphocytosis.

Bacterial Disease: Type # 3. Isolation of the Organism:

1. Culture of blood bone marrow:

Specimens of blood or bone marrow in enteric fever; faeces, vomitus or suspected food in gastroenteritis should be collected before starting treatment.

As there is transient bacteraemia, there are small number of organisms in blood. Repeated culture with larger volume (5-10 ml) of blood is necessary. Blood culture is positive in 80-90% patients in first week and up to 10 days of fever.

The chances of positive culture progressively diminishes with increase in duration of illness. Blood culture also yields positive result during relapses. Bone-marrow material culture is as reliable as blood culture and it yields positive result in 1 to 2 days after commencement of chloramphenicol therapy.

A volume of 5 to 10 ml blood collected aseptically by venepuncture is transferred directly into a blood culture bottle containing 50 to 100 ml 0.5% glucose broth or 0.5% bile broth. Although blood culture in bile broth (selective medium for salmonellae) is most ideal for salmonellae, but, in practice most laboratories use glucose broth for blood culture because all microorganisms including salmonellae grow in the medium.

Larger volume of media helps in dilution of the antibacterial substances present in the blood. Liquid (sodium polyanethol sulphonate) may be added in the media which counteracts the bactericidal action of blood.

Alternatively, 5 ml venous blood, collected aseptically, is allowed to clot in a sterile screw- capped universal container. After removing serum, 15 ml. bile salt streptokinase broth streptokinase, 100 units per ml) is added aseptically to each bottle. The streptokinase digests the clot causing its lysis and thereby the bacteria are released from the clot.

The separated serum is utilised for Widal test. Clot culture offers a higher rate of isolation than blood culture as the bactericidal activity of serum is obviated in the technique.

After incubation overnight at 37°C, sub-culturing is done on MacConkey agar or DCA. Sub-culturing is economically carried out by spreading a loopful of broth over a sector of solid media, one-fourth of an 83 cm Petri dish and incubated for 24-48 hours. Colourless colonies (NLF) appear. Cultures may be discarded as negative after 10 days.

As growth may be delayed and also to eliminate the risk of introducing contamination during repeated subcultures, Castaneda’s method of blood culture may be adopted, which provides both liquid (liver infusion broth) and solid media (3% nutrient agar slope) in one bottle. It is a diphasic medium, broth has an agar slant on one side.

2. Stool and urine culture:

Culture of faeces and urine are always positive during 3rd and 4th week of enteric fever. Repeated cultures are required for positive result. Stool culture is mainly done for detecting the carriers.

3. Duodenal juice or bile culture:

Bile is cultured to detect chronic earners in whom the organisms are present in the biliary tract.

Identification:

In MacConkey’s agar or DCA, salmonellae grow as non-lactose fermenters which are further studied by Gram’s staining, motility preparation and biochemical reactions in different sugar media. Subculture from the colonies is made in nutrient agar to be utilised for agglutination test.

Slide agglutination test:

A loopful isolated orga­nism (unknown culture) from the colony in agar is emulsified in a drop of saline on a microscopic slide without spreading the drop. The emulsion must be absolutely smooth and of medium opacity. One small loopful of specific antiserum is added and then mixed by tilting the slide.

In the same way, a control of bacterial suspension in normal saline is prepared on other slide and to which no specific serum is added. Clumping of bacteria in test slide occurs within a few minutes if the antigen-antibody reaction is specific. Control saline emulsion of bacteria does not show any change.

Biochemically positive strains (Table 12.3) are first tested with polyvalent O serum, which reacts with salmonella strains in groups A to G, provided agglutination is not blocked by Vi antigen, which can be checked by testing all negative strains using S. typhi Vi serum. Positive results with polyvalent O serum indicates a Salmonella strain.

In the next step, the strain is tested with sera prepared against O antigens of the individual salmonella groups. The following are most useful sera which react with factor 2, Group A; factors 4 and 5, group B; with 6 and 7, group C1; with 8, group C2; with 9, group D; and with 3, 10, 15 and 19, group E.

When the strain is positive with individual sal­monella O serum and biochemically it is typically S. typhi, the strain is tested against S. typhi O serum, factor 9. Prompt agglutination indicates that the microorganism belongs to Salmonella group D. Its identity is established as S. typhi by agglutination with salmonella H specific serum which reacts with flagellar antigen d.

In a non-typhoid salmonella, the strain is tested for agglutination with O and H sera for groups A, B and C. When unusual serotypes are encountered, the same should be referred to the National Salmonella Reference Centre (Central Research Institute Kasauli, for human strain, and Indian Veterinary Research Institute, Izatnagar, for salmo­nella of animal origin).

Subtyping:

Subtyping methods are frequently used for the common serotypes (S. typhi, S. typhi- murium and S. enteritidis) which include phage typing, bio-typing, and more recently introduced genotyping methods (plasmid fingerprinting). These techniques are employed in CDC for sub-typing within serotypes of Salmonella.

Serological Test:

1. Widal test:

It is an agglutination test which detects presence of serum agglutinins (H and O) in patient’s serum with typhoid and paratyphoid fever. Salmonella antibody starts appearing in serum at the end of first week and rises sharply during the 3rd week of enteric fever.

It is preferable to test two specimens of sera at an interval of 7 to 10 days to demonstrate a rising antibody titre. Convalescent sera from salmonella gastroenteritis cases often agglutinate a suspension of the causal serotype which helps in retrospective diagnosis.

Procedure:

In Widal test, two types of tubes were originally used:

(i) Dreyer’s tube (narrow tube with conical bottom) for H agglutination and

(ii) Felix tube (short round-bottomed tube) for O aggluti­nation. Nowadays, 3 x 0.5 ml Kahn tubes are used for both types of agglutination.

A serial two-fold dilution of patient’s serum in normal saline (1: 20,1 : 40 and so on up to 1,280 or more) is prepared in 8 small (3 x 0.5 ml) test tubes for each series; 7 for serum dilutions and 8th for a non-serum control.

To the diluted serum and control saline equal volume (0.4 cc) of antigen suspensions (TH, TO, AH and BH) are added and mixed thoroughly by shaking the rack and then the mixtures are incubated at 37°C for 4 hours and read after overnight refrigeration at 4°C. Some workers recommended incubation in a water bath at 37°C overnight.

Observation:

Loose and cotton-woolly clumps are formed in H agglutination and a disc-like granular deposit in O agglutination at the bottom of tube. Con­trol tube shows a compact deposit. The Maximum dilution of serum at which agglutination occurs indicate the titre of antibodies.

The routinely used antigens are H and O of S. typhi, H of S. paratyphi A and B. As paratyphoid O antigen cross reacts with typhoid O antigen due to the sharing of factor 12 by them, paratyphoid O antigens are not used.

Preparation of Widal antigen:

H suspension of bacteria is prepared by adding 0.1% formalin to a 24-hour broth culture or saline suspension of an agar culture. For preparation of O suspension of bacteria, the organisms are cultured on phenol agar (1: 800). Standard smooth strains of the organism are used — S. typhi 901, O and H strains are employed for this purpose.

Interpretation of Widal test:

1. Agglutinin starts appearing in serum by the end of 1st week with sharp rise in 2nd and 3rd week and the titre remains steady till 4th week after which it declines.

2. Rising titre:

Demonstration of rising titre of four-fold or greater of both H and O agglutinins at an interval of 4 to 7 days is the most important diag­nostic criterion.

3. In a single test, a titre of 100 of O or more and a titre of 200 of H agglutinins signifies presence of active infection, but that has to be interpreted taking into consideration the following factors:

(i) Local titre:

Due to sub-clinical infection of salmonellosis in endemic area, low titre of agglu­tinins is present in the serum of normal individuals, which may cause positive reaction. This is known as local titre. Local titre is up to 80 in Kolkata and 60 in Siliguri.

(ii) Immunization:

In immunization with TAB vaccine, vaccinated individuals may show high titres of antibody (H antibody titre 160 or more) to each of the salmonellae.

(iii) Anamnestic reaction:

Persons who had past enteric infection or who have been vaccinated may develop transient fever like malaria, influenza, etc.

(iv) Non-specific antigens (e.g. fimbrial antigen) may produce false-positive result.

(v) Antibiotic treatment:

When treatment with chloramphenicol is started before the appearance of agglutinins, they are unlikely to appear subse­quently; if the antibody is already present, no further rise in titre is expected.

(vi) Carriers:

Widal tests may be positive in many healthy carriers and some have to be detected by Vi antiserum.

2. Other serological tests:

ELISA is sensitive method of measuring antibody against the lipopolysaccharide of Salmonellae, titre of IgM antibody corresponds fairly well with the Widal O titre.

Bacterial Disease: Type # 4. Bacillary Dysentery:

To confirm diagnosis, Shigella (Table 12.4 on pre­vious page) must be isolated in pure culture from an adequate sample. Specimens include fresh stool, mucous flakes, and rectal swabs.

A. Microscopical examination of stool:

Cover-slip preparation in saline and iodine shows large number of pus cells (neutrophils) with dege­nerated nuclei, RBC, and macrophages. The presence of protozoa, cysts or helminthic ova is excluded. The normal bacterial flora is considerably diminished. Fluorescent antibody technique is of some help in rapid diagnosis of a case but it is not usually practiced.

B. Bacteriological examination:

I. Culture:

Media:

MacConkey’s agar, DCA, S-S agar, Selenite-F broth.

A loopful of material is inoculated on selective media like MacConkey’s agar or S. S. agar media.

Selenite-F broth (0.4%) is used as enrichment and transport medium which permits rapid growth of enteric pathogens while temporarily (for 9-12 hours) inhibiting the growth of E. coli. Organisms from selenite-F broth are sub-cultured in MacConkey’s agar after 24 hours incubation at 37°C.

Colourless colonies appear on MacConkey’s agar medium after 12 to 18 hours incubation which are further tested by smear examination, motility preparation and biochemical reactions. Shigellae are gram-negative, non-motile bacilli.

II. Biochemical reactions:

Urease, citrate, H₂S and KCN negative; indole and M R positive organism is suggestive of a shigella strain. S. sonnei is a late lactose fermenter.

III. Slide agglutination test:

It is done by using polyvalent antisera of three groups (A, B and C) of shigella and antiserum of group D (S. sonnei), one by one against the isolate. Monovalent antisera are used for the group for which agglutination has occurred with polyvalent antisera. Then type specific antisera for strains be­longing to group A, B or C is used for agglutina­tion test.

Occasionally, agglutination may not occur due to masking of O antigen by K antigen, which can be removed by boiling the bacterial suspension at 100°C for 60 minutes.

IV. Colicin typing:

It is done for group D strains.

V. Sereny test may be done to confirm the inva­siveness of the isolated strain of shigella, although, it is seldom practiced.

Bacterial Disease: Type # 5. Diarrhoeal Diseases:

Aetiology of infective diarrhoea:

Bacterial diarrhoea can be divided into two groups, those caused by invasive and those caused by non-invasive microorganisms:

(A) Non-invasive bacterial pathogens:

Gastro­enteritis or food poisoning caused by the under-­mentioned organisms are toxin-mediated (toxin type):

1. V. cholerae.

2. Enteropathogenic and entero-toxigenic Esch. coli (EPEC, ETEC).

3. Verotoxin-producing Esch. coli (VTEC E. coli O157: H7).

4. Clostridium perfringens type A.

5. Staphylococcus aureus (some strains)

6. Bacillus cereus.

7. Clostridium difficile.

8. Shigella dysenteriae -1.

9. Vibrio parahaemolyticus.

B. Invasive bacterial pathogens:

These organi­sms cause gastroenteritis or food poisoning by invasive mechanism and not by toxin (non-toxin type):

1. Salmonella species.

2. Campylobacter species.

3. Shigella species.

4. Enteroinvasive E. coli (EIEC).

5. Enterohaemorrhagic E. coli (EHEC).

6. Yersinia enterocolitica.

7. Yersinia pseudo tuberculosis.

A simplified flow chart is shown in Fig. 12.2 out­lining the important steps of examination.

A. Specimens:

1. Faecal sample is examined for detection of the offending agent.

2. Enzyme immunoassay (EIA) of faecal samples is useful in rapid detection of organisms.

3. Blood cultures are indicated only with consti­tutional symptoms.

B. Microscopy:

Saline and iodine preparations when examined microscopically, causative parasitic agents (E. histolytica, Giardia lamblia, and Crypto­sporidium) can be detected.

C. Culture:

Specimen is inoculated in Mac­Conkey’s agar (or in TCBS, thiosulphate-citrate- sucrose-bile salt agar in suspected cases of cholera) and incubated at 37°C overnight.

D. Identification:

Isolated organism is identified by biochemical and agglutination test.

Bacterial Disease: Type # 6. Bacterial Food Poisoning:

Important pathogens are listed in Table 12.5.

1. Isolation of bacteria:

Faeces, and remnant food stuff may be inoculated in blood agar and Robertson’s cooked meat medium (RCM).

2. Detection of toxin in specimen of suspected food and in patient’s serum may be made by toxin-antitoxin neutralisation in mice.

Non-Bacterial Infective Diarrhoea:

Causative agents are shown in Table 12.6:

Specimen:

Faeces.

1. Microscopy:

Saline and iodine preparation of faeces are examined microscopically which will reveal cysts and trophozoites of E. histolytica, cysts of Giardia lamblia and oocysts of Cryptosporidium.

2. Immunoassays and gene probes help to detect viruses.

3. Serological tests:

ELISA of serum is useful.

Bacterial Disease: Type # 7. Cholera:

Vibrio species associated with human infections are shown in Table 12.7:

Specimens:

1. Watery stool and mucous flakes from stool.

2. Rectal swabs from contact and carriers.

Faecal samples are the best source of culture material.

Transport media:

If there are chances of delay, 2-3 gms of faeces are emulsified in 10 to 15 ml V R fluid medium or in bile peptone transport medium or in Cary-Blair medium.

Microscopy:

Rapid diagnosis by direct fluorescent-antibody staining of smears from liquid stool has been used for identifying V. cholerae 01.

Culture:

1. Stool sample is directly plated to one or more selective media like BSA, TCBS, or Monsur’s GTTA medium.

2. The specimens received on holding transport medium are first inoculated into enrichment media such as alkaline peptone water and incubated for 6-8 hours before plating on a selective medium.

Plating media:

Thiosulphate-citrate-bile salt-sucrose agar (TCBS) (Fig. 12.3) and bile salt agar (BSA) are more frequently used plating media. The inoculated plates are generally examined after over­night incubation at 37°C. On TCBS medium, V. cholerae strains appear as yellow sucrose fer­menting colonies. The colonies are moist, transluscent, round, about 1-2 mm in diameter, with a bluish tinge in transmitted light.

Identification:

1. Vibrios are oxidase-positive and ferment glucose which distinguish them from the family Enterobacteriaceae. Vibrios should be differentiated from other related genera — Aeromonas and Plesiomonas.

2. Direct detection of V. cholerae by FAT or co-agglutination test if facilities are existing.

3. Culture and agglutination and biochemical tests.

Biochemical tests:

The El tor biotype is usually haemolytic, V.P. positive, haem-agglutinate chick red cells and resistant to polymyxin B and group IV cholera phage (Table 12.8):

Agglutination test:

Colonies from selective media are picked up with a straight wire and tested with V. cholerae 01 anti­serum. If positive, the test is repeated using mono-specific Ogawa and Inaba sera for serotyping. Hikojima strains react equally well with Ogawa and Inaba sera.

When the agglutination test becomes negative with one colony, the test is to be repeated with at least 5 colonies as V. cholerae-01 and non-01 V. cholerae may coexist in the same specimen.

When the isolated strain of organisms are not agglutinated by V. cholerae-01 antiserum, it should be tested with other O antiserum to establish their identity as belonging to one of 02-0139 serogroups.

Any V. cholerae or suspected to be V. cholerae should be sent to State Reference Laboratory (Cholerae Research Centre, Beliaghata, Kolkata) for confirmation and phage typing.

Serological tests:

It is of little use in diagnosis of cholera but of great help in assessing the incidence of endemic foci. O suspension of cholera vibrios are used for agglutination, indirect haemagglutination, vibriocidal tests and antitoxin assay of serum.

Detection of carriers:

(i) Repeated stool culture is necessary as the vibrio is excreted intermittently.

(ii) It gives better result if stool is collected after administration of a purgative (magnesium sulphate 15-30 g or mannitol 30 g). Bile may be examined after duodenal intubation.

(iii) Serological test is useful in detecting chronic carriers.

Bacterial Disease: Type # 8. Urinary Tract Infections:

Important causative agents of urinary tract infection are listed in Table 12.9:

Laboratory diagnosis:

Pre-therapy culture of urine is necessary for iden­tification of the organism, antimicrobial susce­ptibility test and assessment of the results of treat­ment.

Specimen collection:

Specimen of urine should be adequate and free from urethral or genital tract contamination:

1. Mid-stream clean-catch technique (MSU):

Before collecting a sample, the urethral meatus and adjoining areas should be cleaned with soap and water and then dried with a sterile sponge. The patient begins to pass urine and then mid-portion of the stream is collected in sterile wide-mouthed container or test tube.

2. Catheter specimen (CSU):

In catheterized pa­tients, urine should be collected directly from the catheter and not from the collection bag. The catheter should not be allowed to touch the container.

3. Urine specimens from infants:

A clean catch specimen after careful cleansing of genitalia is pre­ferred.

Although a catheter specimen of urine yields excellent result, but catheterisation to obtain urine is not justified because of the risk of introducing infection.

Transport:

It is important that specimens after collection should reach the laboratory with the minimum of delay, preferably within 2-3 hours after collection or refrigerated at 4°C.

Dip-slide method:

Using dip-slide coated on both sides with culture medium and inoculated by dipping into the freshly passed urine and replacing in a sterile container for transport, which can be read several days after delay.

Specimen is collected in two sterile containers: one for culture and other for routine examination including microscopy.

A. Microscopy:

Pyuria is associated with most clinical infections but may be absent in symptomless bacteriuria. Pus cells along with RBCs are usually found in urinary deposit in haemorrhagic cystitis. Pyuria without bacteriuria may be an indication of renal tuber­culosis.

B. Culture:

1. Media:

(i) Blood agar,

(ii) MacConkey’s agar.

2. Semi-quantitative culture:

Most laboratories use a semi-quantitative technique of inoculation by standard calibrated loop. Dip-slide techniques is useful examining large numbers of urine specimens.

3. Incubation:

After 18-24 hours incubation of inoculated plate at 37°C, discrete colonies develop, each corresponding to the growth of a single bacte­rium.

Semi-quantitative methods:

Calibrated loop method:

A loop delivering 0.001 ml. of urine is used to inoculate blood agar. After incubation of the plate for 18 to 24 hours, plates are examined and colonies are counted.

The total num­ber of bacteria per ml of urine is thus obtained as follows:

Total amount of bacteria per ml of urine – No of colonies x 1,000.

This method is more suitable to study large number of urine samples.

Dip slides:

Commercially available plastic slides coated with CLED (Cysteine lactose electrolyte deficient) agar on one side and MacConkey’s agar on the other side is dipped into the freshly voided urine and replaced in a sterile container and incubated. Viable count is obtained by comparing growth on the media with the manufacturer’s chart.

Although dip slide tech­nique is relatively expensive, it avoids problem of transporting specimens to laboratory particularly from general practitioner’s side, and are useful in screening for significant bacteriuria in busy out­patient department, such as an antenatal clinic.

Interpretation of results:

(i) More than 100,000 (10⁵) viable bacteria of a single species per ml: significant growth, sensitivity test is to be done.

(ii) Between 10,000 to 100,000 bacteria per ml: of doubtful significance, further specimens requires to be cultured.

(iii) Less than 10,000 bacteria per ml: no signi­ficant growth, regarded as contaminants.

4. Identification of the organisms:

The orga­nisms are identified by biochemical and/or sero­logical tests.

Bacterial Disease: Type # 9. Meningitis:

Aetiology:

Most bacteria causing pyogenic meningitis are encapsulated and possess specific affinity for receptors in the choroid plexus or meninges:

1. Common pathogens:

More than 80% infec­tions of pyogenic meningitis beyond neonatal period are caused by:

(i) Streptococcus pneumoniae,

(ii) Neisseria meningitidis, and

(iii) Haemophilus influ­enzae.

2. Infrequent pathogens in remaining 20% cases of pyogenic meningitis include:

(i) Listeria mono­cytogenes,

(ii) Gram-negative enteric rods (e.g. Kleb­siella),

(iii) Staphylococcus aureus,

(iv) Streptococcus pyogenes,

(v) Staphylococcus epidermidis.

Laboratory diagnosis:

Specimen:

CSF is obtained by lumbar puncture. The fluid should be examined immediately after col­lection or placed in the incubator for examination within an hour. The fluid is collected in 3 separate sterile vials — 1 for cell count, 1 for chemical exami­nation, and one for culture.

1. Microscopy:

Gram film of centrifuged deposit shows plenty of pus cells and few Gram-positive or Gram-negative organisms as the case may be (Table 12.10).

2. Chemical analysis:

It shows diminished glucose levels and increased protein (Table 12.10).

3. Culture:

(i) CSF:

Centrifuged deposit is inoculated onto blood agar, chocolate agar and cooked meat broth. The plates are incubated up to 24 hours in an atmo­sphere of 5-10% CO₂.

The isolated organisms are identified by bioche­mical and/or serological tests.

(ii) Blood:

Blood culture is particularly useful in meningitis and may be positive in about 50% of cases of meningitis due to N. meningitidis, H. influ­enzae and S. pneumoniae.

4. Demonstration of bacterial products:

(i) Bacterial antigen can be detected by latex agglu­tination, counter-current immuno-electrophoresis (CIEP)and ELISA using specific antisera to the com­mon serogroups of N. meningitidis serogroup A, serogroup C, H. influenzae and S. pneumoniae.

(ii) Bacterial endotoxin:

The limulus lysate test for detection of bacterial endotoxin is both specific and extremely sensitive.

Bacterial Disease: Type # 10. Tuberculosis:

Classification:

(i) Strict pathogens:

(a) M. tuberculosis complex:

M. tuberculosis — human type

M. bovis — bovine type

M. africanum — human type

M. microti — murine type

(b) Lepra bacilli:

M. leprae — causing leprosy in man

M. leprae murium — causing rat leprosy

(c) Other animal pathogens

M. microti — murine type

M. paratuberculosis — Johne’s bacillus.

(ii) Atypical mycobacteria:

Group I — Photochromogens

Group II — Scotochromogens

Group III — Non-chromogens

Group IV — Rapid growers

(iii) Saprophytic mycobacteria (non-pathogenic):

(a) M. smegmatis — Present in smegma, thermophiles (grow at 52°C).

(b) M. phlei — Present in grass

(c) M. stercoris — Present in dung

(d) M. thermoresistible.

M. tuberculosis complex and M. leprae are found in diseased tissue of humans and other warm-­blooded animals. Most other species (saprophytes and atypical bacilli) are free-living in soil and water.

Laboratory diagnosis:

The diagnosis may be strongly suspected clinically when typical cavitary or calcified lesions are detected radio-graphically, but confirmation requires isolation of bacilli from the lesions.

Important diagnostic tests are shown in Box 12.1:

Specimen:

Specimen is collected from the suspe­cted site of lesion.

1. Pulmonary tuberculosis: Sputum—early morning respiratory specimens of 3 conse­cutive days.

2. Meningitis: CSF.

3. Bone and joints: Aspirated fluid.

4. Renal: Three consecutive morning samples of urine.

Microscopy:

Finding acid-fast slender red rods in sputum or any other adequate sample identifies Mycobacterium as the cause of the disease.

1. Ziehl-Neelsen staining:

Smears made from the specimen in new glass slides is stained by Ziehl- Neelsen method. The acid-fast bacilli appear as pink brightened rods.

Care must be taken in urine sample to exclude commensal smegma bacillus which is only acid-fast but not alcohol-fast whereas tubercle bacilli are both acid and alcohol-fast. It has been observed that there should be 10,000 to 20,000 bacilli per ml of sputum for positive microscopic finding.

2. Fluorescent staining:

Where large number of smears are to be examined, smears stained by fluorescent stains such as auramine phenol or auramine rhodamine are examined under ultraviolet light.

The bacilli appear as bright rods against dark background. Approximately 35-50% of all culture-positive specimens have bacilli detected by acid-fast micro­scopy. Sensitivity of microscopy is high with respi­ratory specimens, particularly with cavitation.

Concentration of specimens:

The bacteria are decontaminated and concentrated in a small volume without inactivation. Such concentrate is mostly used for culture and animal inoculation test and sometime for smear examination.

Several methods of concentration of specimen are in use:

1. Petroff’s method:

It is a simple and widely used technique. Sputum mixed with equal volume of 4% sodium hydroxide is incubated at 37°C for about 30 minutes. The mix­ture is frequently shaken till it gets liquefied and becomes clear. Sodium hydroxide also kills the con­taminating bacteria.

After centrifugation at 3,000 rpm for 30 minutes, it is neutralized with 8% hy­drochloric acid in presence of a drop of phenol red indicator. The deposit is used for smear, culture and animal inoculation.

2. Homogenization method:

The specimen is treated with equal volume of di­lute acids such as 6% sulphuric acid, 3% hydro­chloric acid or 5% oxalic acid, and then clearing the acid by repeated washing with sterile normal saline. The concentration of acid is reduced to half by mixing equal volume of specimen with equal vol­ume of dilute acid.

3. Trisodium phosphate method:

Composition:

(i) 2.94% Trisodium citrate, 3H₂O (0.1M) — 50 ml

(ii) 4% sodium hydroxide (1N) — 50 ml

(iii) N-acetyl-L-cysteine (NALC) — 0.5 gm.

At first mix, trisod citrate with sodium hydro­xide solution. NALC is added to that solution just before use. NALC is a mucolytic agent. 4-6% sodium hydroxide (toxic agent) may kill or seriously injure mycobacteria. The NaOH concentration becomes 2% after mixing equal parts with the specimen.

Principle:

NALC, a mucolytic agent without antibacterial activity, liquefies mucus by splitting disulphide bonds. The mycobacteria are released when the mucous is liquefied.

Method:

1. Add NALC digestant mixture to equal volume of specimen (e.g. sputum, urine, etc) in 50 ml plas­tic centrifuge tubes. Approximately 10-15 ml sample is processed in each tube. The screw cap of the centrifuge tubes are tightened.

2. Homogenise the mixture with a vortex mixture for approximately a slow count of 15, or for a maxi­mum of 30 seconds.

3. Let the tubes stand at room temperature for 15-20 minutes. The specimen should not be exposed to the digestant for more than 20 minutes, because “over-treated” specimens result in fewer positive cultures.

4. After the digestion-decontamination step, add enough phosphate buffer (pH 6.8) (preferred over sterile water) to reach within 1 cm of the top, screw the cap till tightly closed, and invert the tube to mix the solutions and stop the digestion process.

Phosphate buffer acts in the following ways:

(a) It makes strong shifts in pH less likely,

(b) Serves to wash the specimen,

(c) To dilute and neutralize toxic substances,

(d) To reduce specific gravity of the specimen so that sedimentation of the bacilli dur­ing centrifugation is more effective.

5. Centrifuge all tubes at 3,600 x g for 15-20 min­utes.

6. Carefully decant the supernatant into a splash- proof can containing a phenolic disinfectant. Carefully wipe the tip of the tube with cotton soaked with 5% phenol.

7. Re-suspend the sediment with a Pasteur pipette in 1-2 ml sterile water or phosphate buffer (pH 6.8).

8. Inoculate the culture media (LJ slants and Middle-brook 7H10 or 7H11 agar plates) and prepare smear on clean microscope slides for Ziehl-Neelsen or fluorochrome staining.

Culture:

Culture is a more sensitive method for detection of bacilli and, moreover, it is necessary for antimi­crobial drug sensitivity testing.

Inoculation:

Concentrate of sputum and urine are cultured in two bottles of Lowenstein-Jensen medium. It is ideal to inoculate into one egg-based (e.g. LJ) and one agar-based (e.g. Middle-brook) media. Specimens like CSF and tissue biopsies do not require decontami­nation before inoculation as they are unlikely to be contaminated. Bone and joint samples are first dis­rupted in a sterile grinder before inoculation.

Incubaction:

The media are incubated at 36°C-37°C in the dark and light up to 12 weeks. Cultures are examined first after 4 days for rapid growing mycobacteria and then weekly till 8 weeks. The tubercle bacilli usually grow in 2 to 8 weeks. Longer incubation is neces­sary for strains originating from treated patients.

Identification:

The colonies of M. tuberculosis on LJ medium are dry, rough, creamy or buff-coloured (Fig. 12.6) (Table 12.12). Smears from colonies stained by acid- fast stain show acid-fast bacilli (red bacilli) (Fig. 12.7). Growth from LJ medium is further processed for differentiation of mycobacteria (Fig. 12.4).

When an acid-fast bacilli is slow-growing, non-pigmented and niacin positive, it is regarded as M. tuberculosis. Differentiation of strains is done by animal inocu­lation and biochemical tests.

Use of broth culture medium:

It generally takes a long time to grow mycobac­teria in LJ or Middle-brook agar media (Fig. 12.5), particularly in cases of M. tuberculosis, M. bovis, M. avium-intracellular and other slow-growing spe­cies.

However, this time has been shortened through the use of specially formulated broth culture (e.g. BACTEC 12B broth culture) based on radiometric monitoring that support the rapid growth of most of the mycobacteria. Thus, there is a greater rate of recovery of M. tuberculosis and other Mycobacterium species, and the average time of growth has been decreased from 3 to 4 weeks to 10 to 14 days.

Animal inoculation:

Part of the concentrated material (0.5 ml) or the same volume of bacterial suspension from the colonies on culture media is inoculated intramuscularly into the thigh of a tuberculin negative healthy guinea pig of 12 weeks old. Inoculation by subcutaneous route is avoided as it causes a local ulcer which may be infectious.

The animal is weighed prior to inoculation and thereafter at weekly intervals. It is tuberculin-tested after 3-4 weeks and autopsied after 6 weeks. The tuberculin-test becomes positive in infected animal and there is progressive loss of weight.

Autopsy shows:

1. Caseous lesion at the site of inoculation.

2. Enlarged caseous inguinal lymph node. The in­fection may spread to lumbar, portal mediastinal and cervical lymph nodes depending on the severity of the disease.

3. Tubercles may be seen in lungs.

4. Spleen—enlarged with irregular necrotic foci.

5. Peritoneum may show few tubercles.

6. Kidneys unaffected.

Generally, M. tuberculosis is highly infectious for guinea pigs and hamsters and virtually non-patho­genic for rabbits, while M. bovis is highly pathogenic for both rabbits and guinea pigs. “Asian type” of tubercle bacilli and catalase negative and INH- resistant strains of M. bovis are less virulent and do not produce progressive disease in guinea pigs.

Sensitivity testing:

The isolated tubercle bacilli is tested for drug sen­sitivity in LJ medium or Middle-brook agar medium after incorporating different concentrations of anti- tubercular drugs in the media before inoculation (Table 12.13).

More rapid susceptibility testing is achieved by using broth culture medium (BACTEC radiometric system). A rpo B gene has been detected in myco­bacteria by genetic probe (PCR) in mutation induced rifampicin resistance.