A 12 year old girl with high fever reports to hospital ENT OPD with complaint of severe sore throat and difficulty in swallowing food. On clinical examination the pharynx was inflamed with yellowish discharge were over the tonsils.

What is your diagnosis?
It is a case of acute pharyngitis or sore throat.

What are the etiological agents of pharyngitis?
Pharyngitis can be caused by bacteria such as Group A Streptococci, Staphylococcus aureus, Corynebacterium diphtheriae, fusobacterium-treponemes or by viruses such as Herpes Simplex, Epstein Barr virus or even fungus such as Candia albicans.

How is the specimen collected?
Two sets of throat swabs are collected by rubbing over the inflamed area in throat and tonsils. Care should be taken not to touch the tongue, cheeks or palate.

Which transport medium can be used in case of delay?
Pike's medium

How is the specimen processed?
One swab is used for preparing a smear and stained by Gram stain. This will give information on possible etiology. Presence of Corynebacterium diphtheriae can be ruled out by Albert's stain. The other swab is used to inoculate on blood agar and incubated at 37^oC aerobically or in candle jar overnight. Rapid detection of Streptococcus antigens from the throat swab extracts is now possible using latex agglutination, co-agglutination, Enzyme immunoassay  and immunoblot techniques.

How do you identify the growth?
Small, circular, non-pigmented greyish colonies with wide zone of beta hemolysis are seen on Blood agar. Gram stained smear will demonstrate gram positive cocci in chains. A negative catalase test will confirm it as Streptococci. Further identification of the species can be made by Lancefield typing of the isolate. Streptococcus pyogenes reacts with group A antiserum. S. pyogenes is also identified by positive PYR test.

Which are the possible complications of this condition?
Infection may spread by extension locally and result in tonsillar abscess, retropharyngeal abscess , mastoiditis, sinusitis, or otitis media. It may also lead to post streptococcal sequelae.

How would you treat this condition?
S. pyogenes is susceptible to penicillin and cepahlosporins. Patients allergic to penicllin may be treated by other antibiotics such as erythromycin.

Which are the other lesions produced by beta hemolytic streptococci?
Beta hemolytic streptococci (Group A) are responsible for scarlet fever, pyoderma (impetigo), erysipelas, cellulitis, myositis, puerperal sepsis, wound infections, etc. Important post-streptococcal sequelae include Acute rheumatic fever (ARF) and acute glomerulonephritis (AGN).

What is the pathogenesis of ARF?
Recurrent attacks of pharyngitis by Group A Streptococci may lead to ARF. Some degree of antigenic cross-reactions is known to occur between human antigens and streptococcal antigens. An immune response against the bacterial antigens is thought to mediate an attack on cross-reacting self-antigens. Some amount of genetic predisposition is also known to occur in such patients. Certain rheumatogenic strains belonging to M serotypes 1, 3, 5, 6 and 18 are frequently associated with ARF.

How is the diagnosis of ARF made using laboratory tests?
A culture from throat swab is not useful as this condition sets in after an episode of pharyngitis. A retrospective diagnosis can be made serologically by detecting anti-streptolysin O antibodies in patient's serum. The ASO test that is frequently used is based on latex agglutination. A titre of 200 units or higher is considered significant.

Why is anti-streptolysin S not used?
Streptolysin S is not antigenic; hence there are no anti-streptolysin S antibodies.

What is the pathogenesis of AGN?
AGN may follow either pharyngitis or pyoderma by Group A Streptococci. The antibodies that are formed in the body towards streptococcal antigens form antigen-antibody complexes. These complexes get deposited in the glomerular basement membrane of the kidney and elicit complement-mediated attack. Complement levels are usually low in AGN. Certain nephritogenic strains belonging to serotype 1, 4, 6, 12, 25 are associated with pharyngitis-associated AGN whereas serotypes 2, 49, 53, 55, 56, 57, 60 are associated with pyoderma-associated AGN.

How is the diagnosis of AGN made with laboratory tests?
ASO tests are usually not positive in most cases of AGN. Streptodornase test (anti-DNase B) and anti-hyaluronidase tests are more helpful. A titre of 300-350 units or higher of anti-DNase B is considered significant. Streptozyme, a passive hemagglutination test using crude extracts of Streptococci is positive in both AGN and ARF.

A 46 year old febrile man was admitted to the hospital. He had been coughing with yellowish expectoration. Physical examination revealed high fever (38°C), tachycardia, tachypnea and appeared confused. He also complained of chest pain. Breath sound appear crackled. Chest X-ray suggested dense left lower lobe consolidation. Hematological examination revealed leucocytosis and CRP was elevated.

What is your diagnosis?
It is a case of acute lower respiratory tract infection, probably lobar pneumonia. Differential diagnosis must include viral or fungal pneumonia, bronchitis, COPD, lung abscess etc.

Which are the bacterial etiological agents of pneumonia?
Pneumonia can be caused by bacteria such as Streptococcus pneumoniae (pneumococcus), Hemophilus influenzae, Klebsiella pneumoniae, Staphylococcus aureus. Other rarer pathogenic bacteria include Legionella pneumophila and Pseudomonas aeruginosa. People with poor oral hygiene, altered swallowing reflexes, or impaired consciousness are predisposed to infection by anaerobes due to aspiration of oral fluids. Mycoplasma pneumoniae is known to cause primary atypical pneumonia.

What are the specimens collected?
Patient is asked to expectorate sputum into a sterile container. In a severely ill patient specimen such as bronchial washing specimen or transtracheal aspirate may be taken. Blood may be collected for blood culture, CRP & routine blood examination. Urine may also be collected for demonstration of pneumococcal antigens.

How is the sputum specimen processed?
A gram stained smear should be made from the thick part of the sputum and observed for pus cells and bacteria. A good specimen must have less than 10 squamous epithelial cells and more than 25 pus cells per low-power field. The sputum should also be inoculated on to Blood agar or chocolate agar and incubated at 37^oC in 5-10% CO₂, preferably in a candle jar.

What are your observations?
Gram smear of sputum showed plenty of pus cells along with gram positive lanceolate shaped cocci in pairs. Small, circular, smooth, draughtsman-type colonies with alpha hemolysis were seen on blood/chocolate agar.

How do you identify the growth?
Gram stained smear of the colonies revealed gram positive lanceolate shaped cocci in pairs suggestive of pneumococci. These colonies were catalase negative. Bile solubility, inulin fermentation, optochin susceptibility, quellung reaction and mouse intraperitoneal inoculation may be done to differentiate pneumococci from viridans streptococci. Pneumococci are positive for bile solubility, inulin fermentation, quellung reaction, are susceptible to optochin and are pathogenic to mouse. The capsular antgien may be detected by latex agglutination or co-agglutination.

What is quellung reaction?
The Neufeld's quellung reaction is also known as "capsule swelling" reaction. When a suspension of pneumococcal colonies are treated with a loop of serum containing anitbodies to capsular polysaccharide and observed under microscope, the capsule appears swollen. The binding of antibodies to capsular antigen brings about a change in its refractive index, making it appear swollen. A drop of methylene blue may also be added to the suspension to provide contrast. The serum used may be monovalent or polyvalent (omniserum).

What is optochin susceptibility?
Optochin is ethyl hydrocuprein hydrochloride, a disc 5 µg of strength is placed on the lawn culture of pneumococci and incubated. A wide zone of inhibition (at least 10-13 mm diameter) around the disc indicates susceptibility.

What is bile solubility test?
Pneumococci have amidase enzymes that result in autolysis. These enzymes can be activated by surface active agents such as bile salts. Bile solubility test can be done in test tube or in culture plates. To a turbid, 1ml overnight broth culture of pneumococci, addition of few drops of 10% sodium deoxycholate results in clearance of the broth in 15 minutes. Colonies suspected to be of pneumococci are marked and a loopful of 2% sodium deoxycholate is placed on them and incubated at 37^oC for 30 minutes. The disappearance of colonies leaving behind an area of alpha hemolysis indicates positive test.

What is the pathogenesis of pneumococcal pneumonia?
Pneumonia is defined as inflammation and consolidation of the lung tissue due to an infectious agent. Streptococcus pneumoniae reach the lungs after first colonizing the oropharynx. S pneumoniae generally resides in the nasopharynx and is carried asymptomatically in approximately 50% of healthy individuals. Viral infections increase pneumococcal attachment to the receptors on activated respiratory epithelium. Presence of capsule is a major virulence factor as it helps the bacterium to evade phagocytosis. The pneumonic lesion progresses as pneumococci multiply in the alveolus and invade alveolar epithelium. Pneumococci spread from alveolus to alveolus, thereby producing inflammation and consolidation along lobar compartments. A patchy bronchopneumonic pattern involving the lower lobes is seen in the elderly. Since S. pneumoniae infection has a tendency to involve the pleura, pleural effusion is often seen.

Which are the other infections produced by pneumococci?
Pneumococci is known to cause sinusitis, otitis media, bronchitis, lung abscess, septic arthritis, septicemia, meningitis, peritonitis and endocarditis.

Which are the antibiotics used in the treatment of this condition?
Penicillin used to be the drug of choice but large number of strains are now developing resistance due to alteration in the penicillin binding proteins.  Alternate choices include macrolides (erythromycin, roxithromycin, clindamycin), quinolones (ciprofloxacin, levofloxacin) , cephalosporins (cefuroxime, cefpodoxime, cefotaxime). Many of the penicillin-resistant strains are also resistant to erythromycin, cotrimoxazole, tetracycline, and chloramphenicol. The choice of suitable antibiotic must be made after antibiotic susceptibility testing only.

Which conditions can predispose to pneumococcal infections?
Chronic alcoholism, splenectomy, previous viral respiratory illness, malnutrition, chronic smoking, cirrhosis of liver, coronary artery disease etc.

Are any vaccines available against pneumococcal disease?
A 23-valent polysaccharide vaccine against 23 common serotypes has been in use in some countries. 23-valent pneumococcal polysaccharide vaccines has been recommended for use among children aged ≥2 years who have high rates of disease, including those with sickle cell disease (SCD), chronic underlying diseases, human immunodeficiency virus (HIV) infection, or others who are immunocompromised. 23-valent pneumococcal polysaccharide vaccines are effective in preventing invasive pneumococcal disease among older children and adults, these vaccines do not protect children aged <2 years. A 7-valent pneumococcal polysaccharide-protein conjugate vaccine was licensed for use among infants and young children as it decreases colonization and prevents pneumococcal disease among children aged ≤2 years.

A 10 year old child is brought to the hospital with high fever, neck rigidity, and petechial rashes on the body. The child had complained of severe headache and had vomited before being brought in. Photophobia and an altered mental status was also noted. Physical examination showed Kernig's and Brudzinski's signs to be positive.

What is your diagnosis?
It could be a case of acute pyogenic meningitis with septicemia, probably meningococcemia with meningitis.

Which are the bacteria that can cause pyogenic meningitis?
Neisseria meningitidis, Streptococcus pneumoniae, Escherichia coli, Hemophilus influenzae, Streptococcus agalactiae and Listeria monocytogenes can cause acute bacterial meningitis.

What is meningitis?
Meningitis is the term to denote inflammation of the meninges. Depending on the duration of symptoms, meningitis may be classified as acute (hours to days) or chronic (weeks to months). Acute bacterial meningitis is caused by bacteria and is characterized by an acute onset of meningeal symptoms and neutrophilic pleocytosis. Aseptic meningitis characteristically have an acute onset of meningeal symptoms and cerebrospinal pleocytosis that is usually prominently lymphocytic. While viruses cause most cases of aseptic meningitis, it can also be caused by bacterial, fungal, mycobacterial, and parasitic agents.

What is the pathogenesis of meningitis?
Initially, the infectious agent colonizes or establishes a localized infection in the host. This may be in the form of colonization or infection of the skin, nasopharynx, respiratory tract, gastrointestinal tract, or genitourinary tract. Most meningeal pathogens are transmitted through the respiratory route. Both N. meningitidis and S. pneumoniae are known to colonize the oropharynx. From this site, the organism gains access to the CNS by invasion of the bloodstream and subsequent hematogenous seeding of the CNS, a retrograde neuronal (ie, olfactory and peripheral nerves) pathway or direct contiguous spread (ie, sinusitis, otitis media). Once inside the CNS, the infectious agents likely survive because host defenses (eg, immunoglobulins, neutrophils, complement components) appear to be limited. Inflammation of meninges is initiated by the presence of several bacterial components such as lipopolysaccharide and teichoic acid in the subarachnoid space. These components stimulates monocytes and macrophages to produce cytokines such as TNF-α, IL-1 and IL-8. The inflammatory response elicited by these cytokines are responsible for clinical manifestations of meningitis. The fundamental pathologic change in meningococcemia is widespread vascular injury characterized by endothelial necrosis, intraluminal thrombosis, and perivascular hemorrhage. Skin lesions usually contain numerous meningococci undergoing phagocytosis by neutrophils.

How is this condition diagnosed using laboratory techniques?
Since there is both meningitis and septicemia, both blood as well as CSF must be collected. Laboratory diagnosis involves microscopic examination of CSF smear, culture from blood & CSF as well as detection of bacterial antigen.

Which are the specimens collected?
Approximately 3-5 ml Spinal fluid (CSF) is collected by spinal tap (lumbar puncture) by inserting the needle between L3 and L4 vertebrae into a sterile container. Only 3-5 ml of CSF must be collected and the rate of collection should be slow(4-5 drops/second). Alternatively, 1 ml fluid each may be collected in three separate containers. Five ml of venous blood may be drawn by venipuncture for blood culture.

Which are the necessary investigations performed?
CSF should be subjected to cytological (cell type & cell count), biochemical (glucose & protein level) and microbiological investigations. In bacterial meningitis CSF is usually purulent (>100 cells/mm²), containing polymorphonuclear leucocytes and the glucose level is usually less than half the serum level. Microbiological investigations include microscopy (Gram stained smear), antigen detection and culture followed by antibiotic susceptibility testing. If the CSF is clear, it should be centrifuged and the deposit taken for microscopy and culture. A loopful of CSF is inoculated on to Blood agar and Chocolate agar and incubated at 37^oC in the presence of 5-10% CO₂ in a candle jar and incubated overnight. CSF may be subjected to antigen detection by latex agglutination, co-agglutination or counterimmuno-electrophoresis. The blood may be subjected to antigen detection and culture. Blood is inoculated into brain heart infusion broth and incubated  at 37^oC, it is then subcultured to Blood agar the following day.

What are your observations?
The gram stained smear shows plenty of pus cells and gram negative diplococci (both intracellular and extracellular). Round, smooth, moist, glistening, convex, greyish and unpigmented colonies with an entire edge that may be 1-4 mm wide are seen on blood agar. Older cultures may sometimes cause the underlying agar to turn dark. The gram stain of the colonies display gram negative cocci in pairs. These colonies are oxidase positive. Similar colonies are obtained from subculture of blood. The isolate fermented glucose and maltose but not lactose or sucrose. The isolate is identified as Neisseria meningitidis. Latex agglutination test for Neisseria antigen in CSF and blood was also positive.

Meningococcal disease can be treated using antibiotics such as Penicillin and ampicillin. However, due to emergence of drug resistance, third generation cephalosporin such as ceftriaxone is now preferred. MPSV4, a tetravalent polysaccharide vaccine containing 50 μg each of purified bacterial capsular polysaccharides (serogroups A,C,Y,W-135) is available as a single-dose (0.5-mL) vaccine. Conjugate vaccines containing oligosaccharide derived from serogroup C capsular polysaccharide, conjugated to nontoxic mutant diphtheria toxin or tetanus toxoid have been developed. MCV4 is a tetravalent meningococcal conjugate vaccine containing 4 μg each of capsular polysaccharide from serogroups A, C, Y, and W-135 conjugated to 48 μg of diphtheria toxoid and is available as 0.5-mL single dose vaccine. Rifampin, ciprofloxacin, and ceftriaxone are effective in reducing nasopharyngeal carriage of N. meningitdis and are all acceptable antimicrobial agents for chemoprophylaxis.

A 23 old female complains of mild fever, increased frequency, urgency and burning micturition. She also reported a sensation of bladder fullness, lower abdominal discomfort and flank pain.

What is your diagnosis?
It could be a case of urinary tract infection. Some of the differential diagnoses include urethritis, PID, endometriosis, vaginitis and renal calculi.

Which are the bacteria that can cause urinary tract infection?
Common uropathogens of community acquire UTI include E.coli, Klebsiella pneumoniae, Proteus sps, and Enterobacter sps. In hospitalized patient, who have a urinary catheter,  Pseudomonas sps, and Enterococci are common pathogens. Staphylococcus saprophyticus is known to cause UTI in sexually active young women.

What is the common source of UTI?
In community acquired UTI, the uropathogens frequently are one's own enteric flora. UTI is more common in women than men due to proximity of anus to vagina and shorter urethra.

How are urinary tract infections classified?
UTI may be community acquired or hospital acquired, lower or upper, ascending  or descending, uncomplicated  or complicated.

How is the sample collected for laboratory diagnosis?
An early morning, freshly voided, clean-catch, mid-stream urine should be collected in a sterile, wide mouthed container after proper anogenital toilette. The external genitilia must be cleansed with mild antiseptic or soap before sample collection to avoid contamination of the urine by normal flora present in this region. In men, the prepuce is retracted and in women, the labia is spread apart and then the middle portion of the urine is collected in the container. The sample must be labeled and sent to the laboratory without delay.

Which are the other techniques to collect urine specimen?
In infants urine flow may be stimulated by tapping just above the pubis with two fingers at one hour after a feed. One tap per second is given for one minute and after an interval of one minute tapping is continued. Under certain conditions, suprapubic aspiration of urine directly from the bladder may be performed. Since this is an invasive technique, it must be performed only when absolutely necessary. Catheterization only for the purpose of collecting urine should be avoided as it may induce infection. In situations where the patient is already catheterized, the urine must not be collected from the bag, instead, it should be aspirated from catheter tube using needle and syringe.

How long can the urine be held before testing?
Ideally, urine must be processed as soon as possible since urine supports growth of bacteria. In case of delay of 1-2 hours the sample may be refrigerated or treated with boric acid at an concentration of 1.8%. Another way of preserving the sample in case of delay is by collecting urine in sterile vacutainer tubes containing boric acid-sodium formate transport medium. Samples that have been processed after a delay of five hours or more do not give reliable results.

Which investigations are performed on urine sample?
Urine wet mount and culture is commonly performed on urine specimen. Wet mount examination is performed to look for pus cells, RBCs and casts. A loopful of well mixed urine placed on the glass slide (without spreading) can be stained by Gram stain and observed. Presence of single bacterium per oil immersion field in such a smear indicates significant bacteriuria. Screening test such as nitrate reduction, dipstick, tetrazolium reduction etc are not specific and are not routinely done. Leukocyte esterase dip test is helpful in detecting pyuria. Qualitative culture technique such as Miles and Misra are too cumbersome to perform for routine diagnosis, hence a semi-quantitative culture is performed by calibrated loop method. A loopful of well-mixed uncentrifuged urine is inoculated on to CLED agar/MacConkey agar and Blood agar without sterilizing the loop in between.

What is significant pyuria?
Presence of at least 1000 pus cells per ml of uncentrifuged urine is significant pyuria. Ordinarily, presence of ≥10 pus cells/HPF in centrifuged urine and ≥5 pus cells in uncentrifuged urine is considered significant. Some authors consider counts as low as 2-5 WBCs /HPF important in a centrifuged specimen in the female with appropriate symptoms. In women, contamination from vagina may introduce large numbers of pus cells into a sample of voided urine. The presence of squamous epithelial cells along with pus cells in the sample is evidence that contamination has occurred and the pus cell count is not significant.

What is significant bacteriuria?
Since normal voided urine tends to get contaminated with normal flora of the distal urethra, differentiation of contamination from urinary tract infection is made by quantifying the bacterial growth. Significant bacteriuria is a concept put forth by Kass EH, who stated that there should be at least 1,00,000 bacteria of single type per ml of urine. This count may not be applicable in all situations. Recent studies suggest that a count of 10² per ml in acutely symptomatic women and a count of 10³ per ml in symptomatic men may be significant. Any growth obtained from urine collected via suprapubic aspiration is significant. Lower counts may be significant when S. aureus is the pathogen.

How is semi-quantitative culture performed?
A loopful of well-mixed uncentrifuged urine is inoculated on the agar medium without sterilizing the loop and incubated at 37^oC overnight. Following incubation, the number of colonies of single type is counted. A bacteriological loop of 3 mm diameter approximately carries 0.001 ml of urine. If this amount of urine gives rise to at least 100 colonies then the numbers of bacteria present in 1 ml can be obtained by multiplying by 1000, i.e 1,00,000 per ml.

What is your observation?
Wet mount of urine shows plenty of pus cells and RBCs but few squamous epithelial cells. More than 100 colonies of pink coloured (lactose fermenting), smooth, low convex, circular colonies of a single type is seen on MacConkey's agar. Gram stained smear of the colony shows gram negative bacilli, hanging drop shows motile bacilli, and catalase test is positive. Results of biochemical reactions are positive indole test, negative urea hydrolysis, negative citrate utilization, positive MR test and negative VP test. TSI agar shows acid slant/acid butt with little gas but no H₂S. The isolate identified as Escherichia coli was obtained in significant count.

What factors must be borne in mind while interpreting urine culture reports?
Urine in the bladder is sterile, small numbers of bacteria get into the urine from the distal part of urethra while voiding. In typical cystitis, most often urine culture are unimicrobial, recovery of more than one type of bacteria in urine indicates contamination. Bacterial counts in the range of 10²-10³ in the absence of pyuria and other symptoms usually indicates contamination. Rarely, mixed infection by more than one type can occur; in such situations a repeat culture with same results is reliable. Significant bacteriuria may not be applicable for suprapubic aspirated urine. Bacterial counts can be lower if the patient is on antibiotics or has consumed large amount of water before voiding urine. Bacterial counts can be higher if there is a long delay between urine collection and culture.

What is sterile pyuria?
Presence of plenty of pus cells in urine but lack of growth on culture is sterile pyuria. The reasons for this condition include recent administration of antibiotics, UTI by a fastidious/auxotropic/anaerobic bacteria, urethritis due to gonococci,  non-gonococcal urethritis (due to Ureaoplasma, Chlamydia, Trichomonas, or viruses) or renal tuberculosis.

What is baceriuria without pyuria?
It is the presence of large numbers of bacteria in the urine but lack of significant numbers of pus cells. This condition is usually seen in pregnant women where there is retention of urine or when voided urine is held for a long time before culture.

How is this condition treated?
Trimethoprim-sulfamethoxazole for 3 days is considered the current standard therapy for bacterial cystitis. Fluoroquinolones such as norfloxacin also works well. Antibiotics should be selected on the basis of susceptibility testing of the isolate.

A 13-year old male patient was admitted to hospital with complaint of bloody diarrhea along with tenesmus, cramping abdominal pain and fever. The amount of feces passed was scanty. The mucus was blood tinged, feces was not foul smelling and was adherent to the container.

What is your diagnosis?
It could be a case of bacillary dysentery.

Which are the pathogens that can cause dysentery?
Among bacteria that can cause dysentery include Shigella sps and Enteroinvasive E. coli. Bloody diarrhea can be caused by Salmonella sps, Campylobacter sps, Enterohemorrhagic E.coli etc. Among protozoa, Entamoeba histolytica and Balantidium coli can cause dysentery. Differential diagnoses should also include Crohn Disease and Ulcerative Colitis.

What are the differences between bacillary and amoebic dysentery?

	Amoebic dysentery	Bacillary dysentry
Number of motions per day	6-8	>10
Amount of feces	copious	scanty
Odour	Offensive	odourless
Colour	Dark red	Bright red
Nature	Blood and mucus mixed with feces	Blood and mucus, little feces
pH	Acid	Alkaline
Consistency	Not adherent to container	Adherent to the bottom of container
RBCs	Clumps	Rouleaux
Macrophages	Very few	Large and numerous
Pus cells	Scanty	Many
Pyknotic bodies	Common	Nil
Charcot Leydon crystals	Present	Absent
Ghost cells	Nil	Numerous

What is the pathogenesis of dysentery?
Shigellosis is basically an enteric disease and the transmission involves feco-oral route. Shigella is transmitted via food, fingers, fomites or flies. Ingestion of small numbers of bacilli (approximately 10²) can initiate infection in a susceptible person. As few as 10 S. dysenteriae bacilli can cause clinical disease, whereas 100-200 bacilli are needed for S. sonnei or S. flexneri infection. Once they survive the gastric juices, they reach large intestine and  penetrate the cells of the epithelial lining. Incubation period can be short (1-7 days) but can be very short in people with achlorhydria. Pathogenesis requires invasion and toxin production. The characteristic virulence factor is encoded on a large (220 kb) plasmid that is responsible for synthesis of polypeptides that cause cytotoxicity. Shigella also produce siderophores that are coded on plasmid can chelate iron from host cells from its protein-bound state. Shiga toxin (Stx) is not essential for virulence of S dysenteriae type 1 but contributes to the severity of dysentery. The chromosomally encoded enterotoxin are are potent cytotoxins that are responsible for many pathogenic features of Shigella infection. Lipopolysaccaharide plays an important role in resistance to nonspecific host defense encountered during tissue invasion. The bacilli multiplies in the epithelia and spreads laterally to adjacent tissue and penetrate lamina propria. Inflammatory response ensues which ultimately leads to capillary thrombosis and sloughing off of epithelium due to necrosis leaving behind superficial ulcers. Some strains produce enterotoxin (Shiga toxin) that has neurotoxic, cytotoxic and enterotoxic action. The accumulation of inflammatory cells leads to the formation of microabscesses, which spread and coalesce to form larger abscesses. Bleeding occurs from superficial ulcerations. Bacterial shedding usually ceases within 4 weeks of the onset of illness. Chronic carriers are extremely rare.

What is the specimen collected and how is the condition diagnosed with the aid of laboratory?
Freshly passed feces is collected in a sterile wide mouthed container, if no feces is available rectal swabs may also be taken. A saline wet mount may be performed to observe pus cells, RBCs and their arrangement, or presence of any parasitic forms. In case of delay, the feces may be transported in transport medium such as Sach's buffered glycerol saline. Direct gram stained smear or hanging drop preparation is not useful. Feces is inoculated on to MacConkey's agar, and on to selective media such as XLD (xylose-lysine-deoxycholate) agar, DCA (deoxycholate citrate agar) or SS (Salmonella-Shigella) Agar and incubated at 37^oC overnight. The sample may also be inoculated into enrichment broth such as selenite F broth and later subcultured on to MacConkey's agar for subsequent overnight incubation. An enzyme immunoassay for Shiga toxin is used to detect S. dysenteriae type 1 in the stool.

What is your observation?
Pale (non-lactose fermenting), smooth, low convex, circular colonies of a single type is seen on MacConkey's agar. Gram stained smear of the colony shows gram negative bacilli, hanging drop shows non-motile bacilli, and catalase test is positive. Results of biochemical reactions are negative indole test, negative urea hydrolysis, negative citrate utilization, positive MR test and negative VP test. TSI agar shows alkaline slant/acid butt with no gas or H₂S. The isolate ferments mannitol with acid production. The isolate is identified as Shigella sps other than S. dysenteriae, which does not ferment mannitol.

How do you identify the species?
Sigella dysenteriae (Group A) does not ferment mannitol while the rest three serogroups ferment. Shigella sonnei (group D) is a late lactose fermenter and can be identified by a positive ONPG test. It is difficult to differentiate S. flexineri (group B) from S. boydii (group C). Confirmation of their identities can be made by slide agglutination using specific antisera.

Is it necessary to identify the species?
Identity of the species is not important for treatment. S. dysenteriae is known to cause severe form of dysentery than other species because it produces shiga toxin. Species identification is also epidemiologically significant. it is believed that S. dysenteriae is common in underdeveloped countries, S. flexineri in developing countries and S. sonnei is developed countries.

How is Shigella classified?
Shigella are grouped into 4 species: Shigella dysenteriae, Shigella flexneri, Shigella boydii, and Shigella sonnei, also known as groups A, B, C, and D, respectively. Group A has 13 serotypes, group B has 6 serotypes, group C has 18 serotypes, and group D has 1 serotype.

Which are the pathogenicity tests?
Pathogenicity tests to demonstrate Shigella's invasiveness include Sereny's test (development of keratoconjunctivitis in guinea pig), penetration of HeLa/Hep-2 cells in vitro or congo red binding test.

What are the complications of Shigella dysentery?
Complications include bacteremia, hemolytic uremic syndrome (microangiopathic hemolytic anemia, thrombocytopenia, and renal failure), septic arthritis, toxic neuritis, conjunctivitis, intussusception in children, intestinal perforation, and septicemia.

Is antibiotic treatment necessary?
Mild cases of dysentery are self limiting, but in pediatric cases or severe dysentery antibiotic administration is necessary. Resistance of Shigella species to sulfonamides, tetracyclines, ampicillin, and trimethoprim-sulfamethoxazole (TMP-SMX) has been reported worldwide, and these agents are not recommended as empirical therapy. Since Shigella are acquiring resistance to multiple antibiotics, antibiotic susceptibility testing must be performed along with culture.

Several students of a primary school in a village fell ill. All of them were admitted to local hospital following vomiting and diarrhea. Purging was effortless and the feces was non-odorant and rice-watery.

What is your diagnosis?
It is a case of gastoenteritis, probably cholera.

Which are the pathogens that can cause gastroenteritis?
Common bacteria that can cause gastroenteritis include vibrio cholerae and Enterotoxigenic E.coli (ETEC).

What is the pathogenesis of cholera?
Cholera is basically an enteric disease and the transmission involves feco-oral route. Vibio cholerae is transmitted following ingestion of contaminated food or water. Ingestion of large numbers of bacilli (approximately 10⁶) can initiate infection in a susceptible person. Once they survive the gastric juices, they reach small intestine and colonize the epithelial lining and produce cholera toxin. They adhere to the intestinal wall mediated by toxin-coregulated pilus (TCP). Hemagglutinin/protease is a zinc-dependent protease, which cleaves the mucin
and fibronectin and may serve to facilitate the spread and excretion of vibrios within the intestine in stools by detaching them from the intestinal walls. Incubation period can be short (1-3 days). Cholera toxin is holotoxin consisting of A subunit and B subunit. The B subunit binds to specific ganglioside receptors (GM1) on the epithelial cells of small intestine and facilitates the entry of A subunit where it activates adenylate cyclase. Stimulation of adenylate cyclase causes an increased production of cAMP, which leads to hypersecretion of water and electrolytes into the lumen and inhibition of sodium reasborption.

What is the specimen collected and how is the condition diagnosed with the aid of laboratory?
Freshly passed feces is collected in a sterile wide mouthed container, if no feces is available rectal swabs may also be taken. In case of delay, the feces may be transported in transport medium such as Cary Blair medium or VR medium. The enrichment medium alkaline peptone water may also be used as transport medium. Direct gram stained smear and hanging drop preparation may be useful. Feces is inoculated on to MacConkey's agar, and on to selective media such as TCBS Agar and incubated at 37^oC overnight. The sample may also be inoculated into enrichment broth such as alkaline peptone water and subcultured after 6 hours of incubation on to MacConkey's agar for subsequent overnight incubation.

What is your observation?
Hanging drop preparation or darkground microscopy shows actively motile (darting-type) bacilli. Vibrio can be detected by immobilization test, wherein addition of vibrio antisera renders the bacilli in feces non-motile. Gram stain of mucus flakes in feces shows several curved, gram negative bacilli. Pale (non-lactose fermenting), smooth, low convex, circular colonies is seen on MacConkey's agar. On further incubation, colonies turned pink due to late lactose fermentation. TCBS agar shows yellow coloured colonies. Surface pellicle is noticed in alkaline peptone water. Gram stained smear of the colony shows curved gram negative bacilli, hanging drop shows actively motile bacilli, catalase test and oxidase test are positive. Results of biochemical reactions are: positive indole test, negative urea hydrolysis, positive citrate utilization, positive MR test and negative VP test. TSI agar shows acid slant/acid butt with no gas or H₂S. The isolate ferments mannose and sucrose with acid production. Other tests that help in identifying Vibrio are string test and nitrosoindole test. The isolate is identified as Vibrio cholerae.

How do you identify the species?
Slide agglutination with antiserum against H antigen confirms that it is a Vibrio sps. Further slide agglutination using O1 antiserum is used to identify V. cholerae O1 from NAG vibrios. Serotyping by slide agglutination is performed to identify Ogawa, Inaba or Hikojima serotypes. Biotyping is undertaken by performing chick RBC agglutination, Hemolysis, VP test, susceptibility to Polymyxin B and Phage IV to differentiate ElTor from Classical biotype.

Is it necessary to identify the serotype and biotype?
Identity of the serotype and biotype is not important for treatment.  Serotyping and biotyping is only epidemiologically significant.

Is antibiotic treatment necessary?
The mainstay of treatment is replacement of water and electrolytic, restoration of acid-base balance; role of antibiotics is secondary and serves to eradicate bacteria.

What are the complications of cholera?
After dehydration, hypoglycemia is the most common lethal complication of cholera in children. Acidosis in cholera is a result of bicarbonate loss in stools and accumulation of lactate. Acidemia occurs when respiratory compensation is unable to sustain a normal blood pH. Hypokalemia results from potassium loss in the stool.