Mucormycosis (previously called zygomycosis) is a serious but rare fungal infection caused by a group of molds called mucormycetes. These molds live throughout the environment. Mucormycosis mainly affects people who have health problems or take medicines that lower the body’s ability to fight germs and sickness. It most commonly affects the sinuses or the lungs after inhaling fungal spores from the air, or the skin after the fungus enters the skin through a cut, burn, or other type of skin injury. However, it can occur in nearly any part of the body.


Rhinocerebral (sinus and brain) mucormycosis is an infection in the sinuses that can spread to the brain. This form of mucormycosis is most common in people with uncontrolled diabetes and in people who have had a kidney transplant.

Pulmonary (lung) mucormycosis is the most common type of mucormycosis in people with cancer and in people who have had an organ transplant or a stem cell transplant.

Gastrointestinal mucormycosis is more common among young children than adults, especially premature and low birth weight infants less than 1 month of age, who have had antibiotics, surgery, or medications that lower the body’s ability to fight germs and sickness.

Cutaneous (skin) mucormycosis: occurs after the fungi enter the body through a break in the skin (for example, after surgery, a burn, or other type of skin trauma). This is the most common form of mucormycosis among people who do not have weakened immune systems.

Disseminated mucormycosis occurs when the infection spreads through the bloodstream to affect another part of the body. The infection most commonly affects the brain, but also can affect other organs such as the spleen, heart, and skin.

Fungal Agents causing mucormycosis

Rhizopus species, Mucor species, Rhizomucor species, Syncephalastrum species, Cunninghamella bertholletiae, Apophysomyces species, and Lichtheimia (formerly Absidia) species.


The symptoms of mucormycosis depend on where in the body the fungus is growing.

Symptoms of rhinocerebral (sinus and brain) mucormycosis include:

• One-sided facial swelling

• Headache

• Nasal or sinus congestion

• Black lesions on nasal bridge or upper inside of mouth that quickly become more severe

• Fever

Symptoms of pulmonary (lung) mucormycosis include:

• Fever

• Cough

• Chest pain

• Shortness of breath

Cutaneous (skin) mucormycosis can look like blisters or ulcers, and the infected area may turn black. Other symptoms include pain, warmth, excessive redness, or swelling around a wound.

Symptoms of gastrointestinal mucormycosis include:

• Abdominal pain

• Nausea and vomiting

• Gastrointestinal bleeding

Disseminated mucormycosis typically occurs in people who are already sick from other medical conditions, so it can be difficult to know which symptoms are related to mucormycosis. Patients with disseminated infection in the brain can develop mental status changes or coma.

People at Risk

Certain groups of people are more likely to get mucormycosis, including people with:

• Diabetes, especially with diabetic ketoacidosis

• Cancer

• Organ transplant

• Stem cell transplant

• Neutropenia

• Long-term corticosteroid use

• Injection drug use

• Too much iron in the body (iron overload or hemochromatosis)

• Skin injury due to surgery, burns, or wounds

• Prematurity and low birthweight (for neonatal gastrointestinal mucormycosis)

Mode of transmission

People get mucormycosis through contact with fungal spores in the environment. For example, the lung or sinus forms of the infection can occur after someone inhales the spores from the air. A skin infection can occur after the fungus enters the skin through a scrape, burn, or other type of skin injury. Mucormycosis can’t spread between people or between people and animals.


Colony morphology & Microscopy image

• A Medical history, symptoms, physical examinations, and laboratory tests when diagnosing mucormycosis.

• Definitive diagnosis of mucormycosis typically requires histopathological evidence or positive culture from a specimen from the site of infection.

KOH Mount

• Tissue biopsy, in which a small sample of affected tissue is analyzed in a laboratory for evidence of mucormycosis under a microscope or in a fungal culture.

• CT scan of lungs, sinuses, or other parts of your body, depending on the location of the suspected infection.

scamming diagram


Mucormycosis is a serious infection and needs to be treated with prescription antifungal medicine, usually amphotericin B, posaconazole, or isavuconazole. These medicines are given through a vein (amphotericin B, posaconazole, isavuconazole) or by mouth (posaconazole, isavuconazole). Other medicines, including fluconazole, voriconazole, and echinocandins, do not work against fungi that cause mucormycosis. Often, mucormycosis requires surgery to cut away the infected tissue.


The overall prognosis depends on several factors, including the rapidity of diagnosis and treatment, the site of infection, and the patient’s underlying conditions and degree of immunosuppression. The overall mortality rate is approximately 50%, although early identification and treatment can lead to better outcomes.


Protect yourself from the environment. It’s important to note that although these actions are recommended, they haven’t been proven to prevent mucormycosis.

• Wear an N95 respirator face mask.

• Avoid direct contact with lots of dust like constructions, water-damaged

buildings and natural disasters.

• Wear shoes, long pants, and a long-sleeved shirt when doing outdoor


• Wear gloves when handling materials such as soil, moss, or manure.

• Clean skin injuries well with soap and water, especially if they have been exposed to soil or dust.

Emerging respiratory viruses, including novel coronavirus (nCoV)

Virus and Transmission

Viruses continue to emerge and pose challenges to public health

  • Some examples of emerging respiratory viruses include:
    • 2002: Severe Acute Respiratory Syndrome corona virus (SARSCoV)
    • 2009: H1N1 influenza
    • 2012: Middle East Respiratory Syndrome coronavirus (MERSCoV)
    • 2019: Novel coronavirus (2019-nCoV)


  • Human health, animal health and the state of ecosystems are inextricably linked to each other.
  • 70-80% of emerging and re-emerging infectious diseases are known to be of zoonotic origin, meaning they can be transmitted between animals and humans
  • Population growth, climate change, increasing urbanization, and international travel and migration all increase the risk for emergence and spread of respiratory pathogens

Corona Virus

Corona virus structure
  • Corona viruses are a large family of viruses that are known to cause illness ranging from the common cold to more severe diseases such as Middle East Respiratory Syndrome (MERS) and Severe Acute Respiratory Syndrome (SARS)
  • A novel, or new, corona virus is called: nCoV

Origin of corona viruses

  • Corona viruses also cause disease in a wide variety of animal species
  • SARS-CoV was transmitted from civet cats to humans in China in 2002
  • And MERS-CoV from dromedary camels to humans in Saudi Arabia in 2012.
  • Several known corona viruses are circulating in animals that have not yet infected humans.
  • A spillover event is when a virus that is circulating in an animal species is found to have been transmitted to human(s)

Human to Human transmission

  • Corona viruses may be transmitted from person to person, particularly if there is close contact, e.g. during provision of clinical care to an infected patient without applying strict hygiene measures

People at risk

  • People in close contact with Animals (e.g. live animal market workers)
  • Family members or health care workers who are caring for a person infected by a new corona virus


  • Wash your hands with soap and water or alcohol-based hand rub
  • Cover your mouth and nose with a medical mask, tissue, or a sleeve or flexed elbow when coughing or sneezing
  • Avoid unprotected close contact with anyone developing cold or flu-like symptoms and seek medical care if you have a fever, cough and difficulty breathing
  • When visiting live markets, avoid direct unprotected contact with live animals and surfaces in contact with animals
  • Cook your food and especially meat thoroughly.

Nipah virus Highlights and Important information

Nipah virus

• Nipah virus infection in humans causes asymptomatic infection (subclinical) to acute respiratory infection and fatal encephalitis.

• Fatality rate (approx.) 40 – 75 %. • Transmitted from o animals (bats or pigs), o contaminated foods o Human to human

• Natural host Fruit bats of the Pteropodidae family.

• Vaccine – Not available.

• Treatment: supportive. Outbreaks

• First recognized in 1999 during an outbreak among pig farmers in, Malaysia.

• Bangladesh in 2001, followed by nearly annual outbreaks.

• Periodically identified in eastern India (Siliguri, India in 2001).

• Regions at risk: Cambodia, Ghana, Indonesia, Madagascar, Philippines, and Thailand. Transmission

• Direct contact with sick pigs or their contaminated tissues; via unprotected exposure to secretions from the pigs, or unprotected contact with the tissue of a sick animal.

• Consumption of fruits or fruit products (such as raw date palm juice) contaminated with urine or saliva from infected fruit bats.

• Human to human transmission among family and care givers of infected patients.

• Through close contact with people’s secretions and excretions. Signs and symptoms

• It ranges from asymptomatic infection to acute respiratory infection (mild, severe), and fatal encephalitis.

• Infected people initially develop o influenza-like symptoms of fever, o headaches, o myalgia (muscle pain), o vomiting, o sore throat.

• This can be followed by dizziness, drowsiness, altered consciousness, and neurological signs that indicate acute encephalitis.

• Atypical pneumonia and severe respiratory problems, including acute respiratory distress.

• Encephalitis and seizures occur in severe cases, progressing to coma within 24 to 48 hours.

• Incubation period (interval from infection to the onset of symptoms) range from 4 to 14 days. (Can be as long as 45 days)

• Survivor from acute encephalitis gets full recovery, but long term neurologic conditions (seizure disorder, personality changes, relapse or delayed onset encephalitis) may occur.

• Fatality rate: 40 – 75 %.



• The main tests used are o Real time polymerase chain reaction (RT-PCR) from bodily fluids and

o Antibody detection via enzyme-linked immunosorbent assay (ELISA).

o Virus isolation by cell culture.



• Treat severe respiratory and neurologic complications.

• Other supportive care.

Natural host:

fruit bats • Fruit bats of the family Pteropodidae – particularly species belonging to the Pteropus genus.

Nipah virus in domestic animals

• Nipah virus can transmitted in pigs and other domestic animals such as horses, goats, sheep, cats and dogs.

• The virus is highly contagious in pigs. Pigs are infectious during the incubation period, which lasts from 4 to 14 days.

• An infected pig can exhibit no symptoms, but some develop acute feverish illness, labored breathing, and neurological symptoms such as trembling, twitching and muscle spasms.

• Mortality is low except in young piglets.

• Nipah virus should be suspected if pigs also have an unusual barking cough or if human cases of encephalitis are present.



• Controlling Nipah virus in pigs.

• No vaccines available against Nipah virus.

• Routine and thorough cleaning and disinfection of pig farms with appropriate detergents may be effective in preventing infection.

• During outbreak; the animal premises should be quarantined immediately.

• Culling of infected animals – with close supervision of burial or incineration of carcasses.

• Restricting or banning the movement of animals from infected farms to other areas can reduce the spread of the disease.



Diphtheria and Elek’s Gel Precipitation test



This presentation is about Diphtheria and Elek get test.

Elek gel precipitation test is Immunodiffusion in agar. It is used to detect toxin produced by Corynebacterium diphtheriae.

Corynebacterium diphtheriae are Gram positive bacilli.

They are Highly pleomorphic organisms with no particular arrangement.

They are having metachromatic granules at their polar regions.

They are non spore forming, and nonmotile bacterium. Corynebacterium diphtheriae is the pathogenic bacterium that causes vaccine preventable childhood disease, called diphtheria.

It is also known as the Klebs-Löffler bacillus, because it was discovered in 1884 by German bacteriologists Edwin Klebs and Friedrich Löffler.

Special stains like Albert’s stain and Ponder’s stain are used to demonstrate the metachromatic granules.

Here, as shown in image, culture isolate is plated in line and then filter paper shocked with anti toxin is placed perpendicular to inoculation line.

If culture isolate is having toxin, then it will make precipitation line as shown in image with black color.

If culture isolate does not have toxin production then there will be no any toxin antitoxin interaction, and no any line of precipitation, as shown in culture line plated on right side of plate.

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Trypanosoma cruzi

Trypanosoma cruzi

Development and life cycle of Trypanosoma cruzi in insect vector reduviid bugs.


Trypanosoma cruzi passes its life cycle in two host;

  1. Human
  2. Vector reduviid bugs, also known as kissing bugs

This video shows the life cycle of trypanosoma cruzi in human host.

Metacyclic trypomastigote form is infective to human, that is found in feces of insect reduviid bug.

reduviid bugs are nocturnal in habitat.

Human gets infection while insect bite by reduviid bug, that is infected with trypanosoma cruzi.

Along with biting and blood meal, Metacyclic trypomastigote form present in feces are deposited on human skin.

Metacyclic trypomastigote form gets entry in to blood, when abraded skin, mucous membranes or conjunctivae become contaminated with reduviid bug’s feces containing infective form of the parasite.

  1. cruzi can also be transmitted by laboratory accidents, blood transfusion, organ transplantation, vertical transmission from mother to child and rarely by contaminated food or drinks.

After entry in to the blood, the parasite invades reticuloendothelial cells (macrophage) and tissues like, muscles, epithelial cells, nervous tissues.

In side macrophage, there is formation of parasitophorous vacuole.

These parasitophorous vacuole fuses with lysosomes. Inside vacuole it transforms into amastigote form.

Followed by rupture of vacuole and release of amastigote in cell cytosol.

Amastigote multiply by binary fission forming a cyst like mass of growth known as pseudocyst.

Many amastigotes within pseudocyst are transformed into motile nonmultiplying trypomastigote forms.

On rupture of the pseudocyst, trypomastigotes are liberated to blood.

Liberated trypomastigotes are of two types:

  1. Slender highly motile forms
  2. Broader less motile forms

Slender highly motile have elongated nucleus, subterminal kinetoplast and short free flagellum. They are invasive form and migrate to many organs, penetrate cells and continue the life cycle.

Broader less motile have oval nucleus, terminal kinetoplast and long free flagellum. They persist in blood and are taken up by insect vector during blood meal.

Further development occurs in vector reduviid bugs.



Chikungunya is a mosquito-borne viral disease first described during an outbreak in southern Tanzania in 1952. It is an RNA virus that belongs to the alphavirus genus of the family Togaviridae. The name “chikungunya” derives from a word in the Kimakonde language, meaning “to become contorted”, and describes the stooped appearance of sufferers with joint pain (arthralgia).

Signs and symptoms

Chikungunya is characterized by an abrupt onset of fever frequently accompanied by joint pain. Other common signs and symptoms include muscle pain, headache, nausea, fatigue and rash. The joint pain is often very debilitating, but usually lasts for a few days or may be prolonged to weeks. Hence the virus can cause acute, subacute or chronic disease.

Most patients recover fully, but in some cases joint pain may persist for several months, or even years. Occasional cases of eye, neurological and heart complications have been reported, as well as gastrointestinal complaints. Serious complications are not common, but in older people, the disease can contribute to the cause of death. Often symptoms in infected individuals are mild and the infection may go unrecognized, or be misdiagnosed in areas where dengue occurs.


Chikungunya has been identified in over 60 countries in Asia, Africa, Europe and the Americas. The virus is transmitted from human to human by the bites of infected female mosquitoes. Most commonly, the mosquitoes involved are Aedes aegypti and Aedes albopictus, two species which can also transmit other mosquito-borne viruses, including dengue. These mosquitoes can be found biting throughout daylight hours, though there may be peaks of activity in the early morning and late afternoon. Both species are found biting outdoors, but Ae. aegypti will also readily feed indoors. After the bite of an infected mosquito, onset of illness occurs usually between 4 and 8 days but can range from 2 to 12 days.


Several methods can be used for diagnosis. Serological tests, such as enzyme-linked immunosorbent assays (ELISA), may confirm the presence of IgM and IgG anti-chikungunya antibodies. IgM antibody levels are highest 3 to 5 weeks after the onset of illness and persist for about 2 months. Samples collected during the first week after the onset of symptoms should be tested by both serological and virological methods (RT-PCR). The virus may be isolated from the blood during the first few days of infection. Various reverse transcriptase–polymerase chain reaction (RT–PCR) methods are available but are of variable sensitivity. Some are suited to clinical diagnosis. RT–PCR products from clinical samples may also be used for genotyping of the virus, allowing comparisons with virus samples from various geographical sources.


There is no specific antiviral drug treatment for chikungunya. Treatment is directed primarily at relieving the symptoms, including the joint pain using anti-pyretics, optimal analgesics and fluids. There is no commercial chikungunya vaccine.

Prevention and control

The proximity of mosquito vector breeding sites to human habitation is a significant risk factor for chikungunya as well as for other diseases that these species transmit. Prevention and control relies heavily on reducing the number of natural and artificial water-filled container habitats that support breeding of the mosquitoes. This requires mobilization of affected communities. During outbreaks, insecticides may be sprayed to kill flying mosquitoes, applied to surfaces in and around containers where the mosquitoes land, and used to treat water in containers to kill the immature larvae. For protection during outbreaks of chikungunya, clothing which minimizes skin exposure to the day-biting vectors is advised. Repellents can be applied to exposed skin or to clothing in strict accordance with product label instructions. Repellents should contain DEET (N, N-diethyl-3-methylbenzamide), IR3535 (3-[N acetyl N-butyl]-aminopropionic acid ethyl ester) or icaridin (1-piperidinecarboxylic acid, 2-(2-hydroxyethyl)-1 ethylpropylester). For those who sleep during the daytime, particularly young children, or sick or older people, insecticide-treated mosquito nets afford good protection. Mosquito coils or other insecticide vaporizers may also reduce indoor biting. Basic precautions should be taken by people travelling to risk areas and these include use of repellents, wearing long sleeves and pants and ensuring rooms are fitted with screens to prevent mosquitoes from entering.

Disease outbreaks

Chikungunya occurs in Africa, Asia and the Indian subcontinent. Human infections in Africa have been at relatively low levels for a number of years, but in 1999–2000 there was a large outbreak in the Democratic Republic of the Congo, and in 2007 there was an outbreak in Gabon. Starting in February 2005, a major outbreak of chikungunya occurred in islands of the Indian Ocean. A large number of imported cases in Europe were associated with this outbreak, mostly in 2006 when the Indian Ocean epidemic was at its peak. A large outbreak of chikungunya in India occurred in 2006 and 2007. Several other countries in South-East Asia were also affected. Since 2005, India, Indonesia, Maldives, Myanmar and Thailand have reported over 1.9 million cases. In 2007 transmission was reported for the first time in Europe, in a localized outbreak in north-eastern Italy. There were 197 cases recorded during this outbreak and it confirmed that mosquito-borne outbreaks by Ae. Albopictus are plausible in Europe. In December 2013, France reported 2 laboratory-confirmed autochthonous cases in the French part of the Caribbean island of St Martin. Since then, local transmission has been confirmed in over 43 countries and territories in the WHO Region of the Americas. This is the first documented outbreak of chikungunya with autochthonous transmission in the Americas. As of April 2015, over 1 379 788 suspected cases of Chikungunya have been recorded in the Caribbean islands, Latin American countries, and the United States of America. 191 deaths have also been attributed to this disease during the same period. Canada, Mexico and USA have also recorded imported cases. On 21 October 2014, France confirmed 4 cases of locally-acquired chikungunya infection in Montpellier, France. In late 2014, outbreaks were reported in the Pacific islands. Currently chikungunya outbreak is ongoing in Cook Islands and Marshall Islands, while the number of cases in American Samoa, French Polynesia, Kiribati and Samoa has reduced. WHO responded to small outbreaks of chikungunya in late 2015 in the city of Dakar, Senegal, and the state of Punjab, India. In the Americas in 2015, 693 489 suspected cases and 37480 confirmed cases of chikungunya were reported to the Pan American Health Organization (PAHO) regional office, of which Colombia bore the biggest burden with 356 079 suspected cases. This was less than in 2014 when more than 1 million suspected cases were reported in the same region. In 2016 there was a total of 349 936 suspected and 146 914 laboratory confirmed cases reported to the PAHO regional office, half the burden compared to the previous year. Countries reporting most cases were Brazil (265 000 suspected cases), Bolivia and Colombia (19 000 suspected cases, respectively). 2016 is the first time that autochthonous transmission of chikungunya was reported in Argentina following an outbreak of more than 1 000 suspected cases. In the African region, Kenya reported an outbreak of chikungunya resulting in more than 1700 suspected cases. In 2017, Pakistan continues to respond to an outbreak which started in 2016.

More about disease vectors

Both Ae. aegypti and Ae. albopictus have been implicated in large outbreaks of chikungunya. Whereas Ae. aegypti is confined within the tropics and sub-tropics, Ae. albopictus also occurs in temperate and even cold temperate regions. In recent decades Ae. albopictus has spread from Asia to become established in areas of Africa, Europe and the Americas. The species Ae. albopictus thrives in a wider range of water-filled breeding sites than Ae. aegypti, including coconut husks, cocoa pods, bamboo stumps, tree holes and rock pools, in addition to artificial containers such as vehicle tyres and saucers beneath plant pots. This diversity of habitats explains the abundance of Ae. albopictus in rural as well as peri-urban areas and shady city parks. Ae. aegypti is more closely associated with human habitation and uses indoor breeding sites, including flower vases, water storage vessels and concrete water tanks in bathrooms, as well as the same artificial outdoor habitats as Ae. albopictus. In Africa several other mosquito vectors have been implicated in disease transmission, including species of the A. furcifer-taylori group and A. luteocephalus. There is evidence that some animals, including non-primates, rodents, birds and small mammals, may act as reservoirs.




A large group of Gram-positive cocci distributed widely in men and animals, mostly forming part of normal flora, but some species responsible for some major infections. Individual cells 0.5 -1 µm in diameter and because they divide in one plane only, occur in pairs and chains. The medically significant streptococci may be conveniently divided on the basis of either hemolysin on blood agar (complete hemolysins – beta, partial hemolysins – alpha. No hemolysins – gamma) or by the presence or absence of a group specific carbohydrate antigen (i.e. Lancefield).


  • Streptococcus pyogenes:


Characteristics: Gram-positive cocci in chains, cells less then 1µm diameter, non-motile, non-sporing.

Transmission: Normal habitat is the human upper respiratory tract and skin. Spread by airborne droplets and by contact. Survival in dust may be important.

Epidemiologic typing of strains based on M and T proteins useful in outbreaks.


  1. Infection of upper respiratory tract
  2. Skin and soft tissue (pharyngitis, cellulitis, erysipelas, lymphadenitis).
  3. Toxic manifestation includes scarlet fever.
  4.    Non-suppurative sequelae:    Acute glomerulonephritis and Rheumatic fever
  5. Pyogenic infection

Laboratory Diagnosis:

Sample collection:

Throat swab, pus or blood


Methods for identification of orgainism:


Streptococcus species are non-motile, non-sporing coccus.


 Direct methods:

  1. Gram staining : Gram-positive cocci, cells often in pairs and chains
  2. Culture: Culture media: Blood agar

Grow well aerobically and anaerobically

       β haemolysis and Bacitracin sensitive

  1. Antibiotic sensitivity test


Indirect methods:


  1. S.O. titer (Anti streptolysin O titer): It is important in the investigation of post streptococcal diseases. Titer more than 200 IU/ml is significant


  1. Estimation of DNAas B antibody: used for diagnosis of post streptococcal Acute glomerulonephritis and Rheumatic fever. Titer more than 300 IU/ml is significant


  • Streptococcus pneumoniae


Characteristics: Gram-positive coccus characteristically appearing in pairs in Gram films. Cells approximately 1 µm in size, often capsulate. Requires blood or serum for growth. Capable of aerobic and anaerobic respiration; growth may be enhanced in CO2.


Diseases: Pneumonia (Capsular type III frequently associated with pneumonia), Septicemia, Meningitis, Bacteraemia, Endocarditis, paricarditi, Otitis and related infection in children.


Transmission: Normal habitat is the human respiratory tract; up to 4 % of population may carry in small numbers. Transmission via droplet spread.


Laboratory Diagnosis:

Sample collection:

  1. Sputum for microscopy and culture.
  2. Blood for culture
  3. C.S.F. for microscopy, culture and biochemistry


Methods for identification of orgainism: non-motile, non-sporing coccus.


 Direct methods :


1.Gram staining :  Gram-positive cocci, diplococcus, capsulated

2.Culture:             Culture media: Chocolate agar, blood agar

Grow well in carbon dioxide enriched media,

                             α haemolysis, draughtsman colonies that may autolyse within 48 h at 350


  1. Biochemical Reactions :


  • Catalase negative.
  • Susceptible to bile and optochin.
  • Polysaccharide capsule can be demonstrated by appropriate staining techniques. They are antigenic and in the presence of specific antiserum appear to swell (quellung reaction).


  1. Antibiotic sensitivity test


  • Enterococcus:


Formerly classified in the genus Streptococcus with which they share many characteristics; there are currently 15 species of which two, E. faecalis and E. faecium are of medical importance and are considered together.


Characteristics: Gram-positive cocci, cells often in pairs and chains; more ovate-appearance than streptococci. Non-fastidious; capable of aerobic and anaerobic respiration.


Diseases:  Urinary tract infection  and wound infection.


Laboratory Diagnosis:


Sample collection:

Throat swab, pus or blood for isolation of S. pyogens

High vaginal swab, blood, CSF., ear swab for isolation of S. agalactiae

Urine or pus to isolate enterococci

Blood for A.S.O. titer


Methods for identification of orgainism:


Streptococcus species are non-motile, non-sporing coccus.


 Direct methods:


  1. Gram staining : Gram-positive cocci, cells often in pairs and chains
  2. Culture: Culture media: Blood agar

Grow well aerobically and anaerobocally,

β or no haemolysis

Bile aesculin test positive

Litmus milk reduction test positive

Heat tolerant, Salt tolerant and Bile tolerant


  1. Antibiotic sensitivity test


viridans Streptococci: 

There are several species of alpha-hemolytic streptococci. Most species are commensals in the mouth. S. mutans is strongly associated with dental carries. Several species are capable of causing bacterial endocarditis.

Diphtheria and Neisseria



Genus Neisseria:


This genus contains several more or less fastidious species of which two, N. gonorrhea and N. meningitides are important pathogens.




Characteristics: Non-motile Gram-negative diplococci with fastidious growth requirements: capnophilics; capsulate.


Diseases:      1)   Meningitis

2)   Septicemia in absence of meningitis

3)   Chronic meningococcal arthritis


Transmission: Human pathogens; no animal reservoir. This carried in pharynx. Carriage rate in population increases during epidemics. Droplet spread N. meningitidis has several immunologically distinct capsular types (A, B, C).


Laboratory Diagnosis


Sample collection:


  1. Cerebrospinal fluid
  2. Blood for culture in septicaemia
  3. Aspirate from skin leasion or pus from infected joint
  4. Swab from nasopharynx and throat to detect carriers

Transportation of sample: Transport sample as early as possible. Never refrigerate the sample. It should be kept at 35º-37ºc


Processing of the sample:


  1. C.S.F. sample is collected in two sterile test tubes- sample no. 1 for culture and sample no. 2 for cell count , microscopy and biochemistry
  2. The smears are prepared directly from the sample if it is turbid.if slight sloudy,centrifuge the sample and sediment is used for preparation of smears and culture


Methods for identification of organism:


 Direct methods:

  1. Gram staining: Gram-negative diplococcic with adjacent sides flattened cells often in pairs and chains and some are seen intracellular in polymorphoneuclear cells.
  2. Culture: Culture media: chocolate agar

Grow well at 37°C in moist atmosphere containing 5-10% CO2

  1. Biochemical reactions: a) Oxidase test positive
  2. b) Sugar fermentation test:

Glucose   –   Acid no gas                      Maltose  –      Acid no gas


  1. Antibiotic sensitivity test


Indirect methods: Slide agglutination test, coaglutination test and latex agglutination test




Characteristics: Non-motile Gram-negative diplococci with fastidious growth requirements: capnophilics; Noncapsulated.


Diseases:     N. gonorrhea:       1)   Gonorrhea,

2)  Pelvic inflammatory disease

3)  Salpingitis in females

4)  Ophthalmia neonatorum in infants


Transmission: Human pathogens; no animal reservoir. This may be carried in genital tract, nasopharynx and anus. Spread by sexual or intimate contact.


Laboratory D1agnosis:


Sample Collection:


  1. Urethral discharge collected by calcium alginate swab
  2. Cervical swab from non-puerperal women


Transportation of sample: Samples are transported in Amies transport medium.


Methods for identification of organism:


 Direct methods:


  1. Gram staining: Gram-negative diplococcic with concave adjacent sides (kidney shape), seen intracellularly  in polymorphoneuclear cells.


  1. Culture: Culture media: Chocolate agar, Modified Thayer martin madium

Grow well at 37 c in moist atmosphere containing 5-10% co2


  1. Biochemical reactions: a) Oxidase test positive
  2.  b)   Sugar fermentation test:

Glucose   –   Acid no gas             Maltose  –      No reaction


  1. Antibiotic sensitivity test


Indirect methods: Direct fluroscent antibody test and Slide agglutination test





This genus contains many species, is widely distributed in nature, and is part of a spectrum, with Mycobacterium and Nocardia, of similar cell wall structure containing mycolic acids. The species of major importance is C. diphtheriae. This and other pathogens within the genus need to be distinguished from commensal corynebacteria.


Corynebacterium diphtheriae


Characteristic: Gram-positive, non-capsulate, non-sporing, non-motile rods, 2-6 µm in length. In Gram-stained films cells arranged as “Chinese letters” or palisades and showing irregular staining or granule formation are characteristic.  Non-fastidious, but growth enhanced by inspissated serum (Loeffler medium). Capable of aerobic and anaerobic respiration.


Transmission: Normal habitat: usually nasopharynx, occasionally skin of humans. Infection is usually spread by aerosol. Patients may carry toxigenic organisms for up to 2-3 months after infection.



Pathogenesis: Diseases is due to production of diphtheria toxin controlled by the tox gene, which is integrated into the bacterial chromosome on a lysogenic phage. When concentration of exogenous inorganic iron (Fe3+) is very low, toxin production is maximal; the selective advantage to the organism is unknown. The mode of action of the toxin is to block protein synthesis of the host cells by inactivating an elongation factor.


Diseases: Diphtheria caused by toxigenic strains of C. diphtheriae. Focus of infection may be the throat or, increasingly commonly the skin.





Commensal Corynebacteria are normally present in the throat, skin, conjunctiva and other area. These may sometimes be mistaken for C.diphtheriae and are called diphtheroids. In general, diphtheroids possess few or no metachromatic granuals and are arranged in palisade (Parallel rows) rather than in Chinese letter pattern. Some diphtheroids are indistinguishable from diphtheria bacilli microscopically and required to be differentiated by biochemical test and more reliably by virulence test.




Sample collection:

  1. Throat and nasopharyngeal swab
  2. skin swab from for diagnosis of cutaneous diphtheria


Methods for identification of orgainism:

Gram-positive, non-capsulate, non-sporing, non-motile rods


 Direct methods:


    1. Gram staining : Gram-positive rods are seen arranged as Chinese letters” or palisade( commansal corynebacteria)


  1. Albert staining: diphtherae stains green and beaded due to the presence of dark staining granules in rods. These granules,known as volutin granules or metachromatic granules, are energy storing inorgainic poly-phosphate units


  1. Methylene blue staining: diphtherae stains unevenly with dark blue staining granules in rods


  1. Culture: Culture media: Loffler’serum medium, Tellurite blood agar  (Black colony)

Grow well aerobically. Temperature range for growth is 20-40ºC


  1. Biochemical reactions:

Sugar fermentation test: ferment Glucose and maltose with production of acid

Catalase positive


  1. Animal inoculation:

        Intracutaneous injection in to guineapig leads to death of the animal.


Indirect methods:

Toxigenicity testing of C.diphtheria by  Elek gel precipitation test and   Schick test




            Mycobacteria are widespread both in the environment and in animals. The major human pathogens are M. tuberculosis and M. leprae, but awareness of the importance of other species is increasing with their recognition as pathogens in AIDS patients.


Characteristics: Aerobic rods with a Gram-positive cell wall structure, but stain with difficulty because of the long-chain fatty acids (mycolic acids) in the cell wall. Acid-fastness can be demonstrated by resistance to decolorization by mineral acid and alcohol (Ziehl-Neelsen stain). Mycobacteria grow more slowly than many other bacteria of medical importance, but the genus can be divided into: rapid growers (form visible colonies within seven days); slow growers (form visible colonies only after 14 days’ incubation).



Transmission: Droplet spread aided by ability of organism to survive in the environment (M. tuberculosis, M. leprae). Milkborne spread of M. tuberculosis to humans from infected cattle has been important in the past. Social and environmental factors and genetic predisposition all have a role. Leprosy requires close and prolonged contact for spread.


Diseases: M. tuberculosis causes tuberculosis in humans and animals. M. leprae is restricted to man and causes leprosy. Mycobacteria other than tuberculosis (MOTT) are associated with a range of conditions, usually in immunocompromised hosts. The avium-intracellulare complex has important association with AIDS patients.




Sample Collection:

  1. Sputum: Spot and the morning sample should be collected in clean, dry, wide necked & leak proof container before any mouthwash is used.

Send it to laboratory within 2 hours or kept at 4ºC until delivery is possible.

  1. Gastric wash: children
  2. Body fluids ( Synovial, Pleural, Pericardial, Ascitic fluid)
  3. CSF
  4. Urine


Conventional Methods:


  • Direct Smear Examination:
    • Z N staining: Acid – fast bacilli are seen
    • Fluorescent Staining


  • Culture:  Early morning 2 sputum samples

Decontamination & concentration


  • Media: ~ Egg-based                 J. medium

~ Agar-based               Middlebrook

~ Incubation                 4-6 weeks            


  • Biochemical Reactions:

Niacin test and Nitrate reduction test are two important tests for identification of M. tuberculosis


  • Tuberculin Test:

It is valuable diagnostic test in paediatric age group.


Newer Methods:


MGIT (Mycobacteria Growth Indicator Tube): Culture tube contains Middle brook 7H9 broth and a fluorescent compound embedded in a silicon sensor. Growth is detected visually using an ultraviolet light. Oxygen diminishes the fluorescent output of the sensor; therefore, Oxygen consumption by organisms present in the medium is detected as an increase in fluorescence.


MODS (Microscopic Observation Drug Sensitivity): It is liquid culture method based on microscopic detection of characteristic mycobacterium tuberculosis morphology. It utilizes the tuberculosis cording phenomenon for identification, which is easily viewed with an inverted microscope. It detects susceptible, mono resistant and multi drug resistant tuberculosis usually within a 5 to 10 days incubation period. It can be used to monitor patient on therapy, detects only living organisms.


LPA (Line Probe Assay): It is based on nucleic acid amplification technology which allows for rapid detection of Mycobacterium tuberculosis complex along with rifampicin resistant alone or in combination with isoniazid resistant in direct sputum sample or in culture isolates.


Gene Expert: It is a cartridge based automated new molecular diagnostic test that detect the presence of Mycobacterium tuberculosis as well as detect resistant to drug rifampicin. It can provide reliable results in about 2 hrs. It detects DNA sequence specific for Mycobacterium tuberculosis and

drug resistant by PCR.


BACTEC: It is radiometric method which detects the growth of mycobacteria in about a week by using 14C labeled substrates. Culture media contains 14C-labelled palmitic acid. Mycobacteria metabolize the 14C-labelled substrates and release radioactively labeled 14CO2. The instrument measures 14CO2 and reports in terms of ‘growth index’. A growth index of ≥10 is considered as positive. This method can also differentiate between M. tuberculosis and M. bovis.


Sensitivity Testing:


BACTEC can also be used for sensitivity testing of antituberculus drugs to identify drug resistance.

Luciferase reporter mycobacteriophage (chemiluminescence) has been used for susceptibility testing of M. tuberculosis.

Epsilometer test (E-Test) has also been applied for susceptibility testing of M. tuberculosis.



  1. Specimen: Nasal mucosa, Skin lesion and ear lobules. Skin specimen are obtained by slit and scrape (edge of the lesion) method
  2. Acid Fast Staining: Smear is stain by Ziehl Neelson Method using 5% H2SO4 as decolourising agent. The Smears are graded, based on the number of the Bacilli as follows:
1-10 Bacilli in 100 fields 1+ 10-100 Bacilli per fields 4+
1-10 Bacilli in 10 fields 2+ 100-1000 Bacilli per fields 5+
1-10 Bacilli per fields 3+ More than 1000 Bacilli 6+


  1. Skin and Nerve Biopsy: Useful in diagnosis and classification of leprosy lesion.
  2. Animal inoculation: Foot pad of mouse and 9 Banded Armadillo are used.
  3. Lepromin Test: This test used to assess the prognosis and response to treatment.

6. Serological Test: By detection of anti-phenolic glycolipid-1 antibodies.various serological test like latex agglutination, ELISA and M. leprae particle agglutination test.

Diagnostic Parasitology




          The following are the main ways in which parasitic infections are diagnosed in the laboratory.


Microscopic examination:  The majority of intestinal, urinary, and blood parasites can be detected microscopically in unstained or stained preparations, either directly or following concentration techniques.


Culture techniques: Only a minority of parasitic infection is diagnosed routinely by culture   techniques.


Immunodiagnosis: With the development of reagents, which are both more sensitive and specific, immunodiagnosis techniques are becoming increasingly used in diagnosis and in studies involving the epidemiology and control of parasitic diseases.




Feces should be examined within a few hours of being passed. If amoebic dysentery is suspected, the specimen should be examined as soon as possible and kept in a warm environment until it is examined.


Gross examination:

Report the appearance of the specimen:

Mention: Color, Consistency, (i.e. whether it is formed, semiformed, unformed, or fluid), whether it contains blood, mucus, pus, whether it contains worms or worm segments.


Microscopic examination:

Place a drop of fresh physiological saline on one end of a slide and a drop of Lugol’ iodine on the other end. Using a piece of stick, mix a small amount of specimen (i.e. matchstick head size) in the saline and iodine. Make wet preparations.


Use of saline and Iodine:

In a saline preparation motile parasites such as amoeba, flagellates, larvae, and ciliates can be identified. Helminth eggs can be readily identified. Cysts can also be detected but they are much more easily seen in iodine prepration because it stain nuclei and glycogen mass.


Examine systematically the entire saline preparation using 10X objective.Use 40X objective to identify small parasites.


Normal structures found in feces.

Care must be taken not to report as parasites those structures, which can be normally found in feces such as muscle fibers, vegetable fibers, starch cells, pollen grains, fatty acid crystals, soaps, spores, yeasts, and hairs.


Concentration techniques for fecal parasites:

Concentration methods to detect parasites in feces may be necessary for the following reasons:

  • To detect parasites when they are not found in a direct examination but the symptoms of intestinal parasitic infection continue.
  • To detect the eggs of parasites, which are often, few in number such as those of Schistosoma or Taenia species.
  • To check whether treatment has been successful.
  • To investigate the prevalence and incidence of parasitic infection as part of an epidemiological survey.



The following techniques are used commonly:


Floatation Technique

  1. Formal ether technique in which parasites are sedimented by centrifugal force. Ether is used to dissolve fecal fat and to separate the fecal matter from the sedimented parasites.
  2. Formal detergent technique in which parasites are sedimented by gravity using a solution of low specific gravity with detergent added to clear the fecal mater.


Concentration Technique:

  1. Sodium chloride floatation technique in which parasites are floated in saturated sodium chloride.
  2. Zinc sulphate floatation technique in which parasites are float in 33% w/v zinc sulphate.


The recommended technique for hospital laboratories is the formal ether technique. It is rapid and gives good concentration of parasitic cysts, eggs, and larvae, in fresh or preserved feces. The formal detergent sedimentation technique is suitable replacement for the formal ether technique if a centrifuge is not available and results are not required urgently. Floatation techniques are not recommended as routine concentration techniques because they concentrate only a small range of fecal parasites.


Examination of Blood:

Examination of blood smear stained with Giemsa stain is the most common method of detecting Plasmodium spp., Babesia spp., Trypanosoma spp., and some species of Microfilariae. Although motile organisms such as Trypanosoma spp. And Microfilaria can be detected on a wet preparation of a fresh blood specimen under low and high power magnification, identification is made on the basis of characteristics seen on a permanently stained smear. Concentration methods using membrane filters can be used to detect Trypanosoma spp. or Microfilariae but are rarely performed in the clinical laboratory.


Collection and preparation of the blood specimen:

Blood taken directly from a finger stick should be used for a malarial smear because it tends to give best staining characteristics. Blood collected in EDTA gives adequate staining if processed within 1 hour. With Giemsa stain cytoplasm of the parasite stains bluish and the chromatin red to purple red. Giemsa staining gives best morphological details but it is a time consuming procedure.


Identification of the organism:

For the suspected case of blood parasites, both a thick and thin film should be made. Both preparations can be made on the same slide or on separate slides.


A thick film is best for detection of parasites, because organisms are concentrated in relatively small area. The thick film is made by pooling several drops of blood on the slide and then spreading it onto a 1.5 cm area. Thickness is optimal when newsprint is barely visible through the drop of blood before it dries. The blood should be allowed to dry for at least 6 hours before staining. It should not be fixed with methanol. Initial scanning of the stained smear at 10X detects microfilariae. At least 100 oil immersion fields should be examined before a negative result is reported.


Species identification should be made from a thin film, because the characteristics of the parasite and the RBC can be seen. The thin film is made in the same way as that for a differential count. It should be fixed in methanol for 1 minute and air dried before staining with Giemsa stain. The entire smear should be scanned at 10X for detection of the large parasites such as microfilariae; then at least 100 oil immersion fields must be examined for the presence of organisms such as Trypanosoma spp. or for intracellular organisms such as Plasmodium spp. or Babesia spp.



Immunodiagnosis of parasitic infection:

Immunodiagnosis is used to assist in the clinical diagnosis of parasitic infection and in the epidemiology and control of parasitic diseases.


Immunodiagnosis techniques are based on the detection of:

  • Antibody in a person’s serum, produced in response to a particular parasitic infection. The antibody may persist for a long period in the serum after an infection has ended and therefore antibody tests are unable to distinguish between a past or present infection.
  • Antigen, which is excreted by parasites and can be found in serum, urine, CSF, feces, or other specimens.


Immunodiagnostic techniques are required when:

  • Parasites live in the tissues of internal organs and cannot therefore be easily removed for examination.
  • Parasites can be found in specimens only in certain stages of an infection.
  • Parasites are present only intermittently or in to few numbers to be easily detected in specimens.
  • The techniques used to detect parasites are complex or time-consuming.



Those parasitic diseases for which Immunodiagnosis is of particular value include:

  • South American trypanosomiasis, chronic stage.
  • African trypanosomiasis, when parasitaemia is low.
  • Visceral leishmaniasis.
  • Cutaneous and mucocutaneous leishmaniasis.
  • Amoebic liver abscess.
  • Hydatid disease.
  • Filariasis, occult and chronic infection.
  • Schistosomiasis, chronic stage.









E. histolytica

Cysts, amoeba

G. lamblia

Cysts, flagellate

B. coli

Cyst, ciliate

I. belli





Egg, segment

H. nana


D. latum


F. hepatica


F. buski




A. lumbricoides

Egg, worm

E. vermicularis


S. stercoralis


T. trichiura


A. duodenale


N. americanus




Trophozoite, schizont, gametocyte



W. bancrofti


B. malayi


L. loa





S. haematobium


S. mansoni


T. vaginalis


W. bancrofti


O. volvulus








Bone marrow

L. donovani


T. gondii


Lymph gland & aspirate



L. donovani


T. gondii


Liver aspirate

E. histolytica


L. donovani


T. gondii


Spleen aspirate

L. donovani


T. gondii





O. volvulus


D. medinensis


E. vermicularis





Duodenal aspirate

G. lamblia


F. hepatica


S. stercoralis


Bronchial biopsy aspirate

P. carinni




Parasites Form Microscopic Features
E.histolytica Amoeba *Average size is about 25*20 µm.

*Active amoeboid movement (directional) in fresh warm


*Contains ingested red cells.

Cyst *Round, measuring 10-15 µm.

*Contains 1, 2, or 4 nuclei.

*Chromatoid bars can be seen in immature cysts.



Flagellate *Pear-shaped, usually measuring 10-12*6 µm.

*Upper end has a concavity with a sucking disc.

*There are 8 flagella.

Cyst *Oval in shape.

*Small, measuring about 10*6 µm.

*Contains the remains of axonemes and parabasal bodies.

*Thread-like remains of flagellate may also be seen.

*4 nuclei.

B.coli Ciliate *Large, measuring 50-200*40-70 µm.

*Rapid revolving motility.

*Beating cilia can be seen, especially around the cytostome.

Cyst *Large, measuring 50-60 µm.


*Cilia may be seen in younger cysts.

*Macronucleus visible.

I.belli Oocysts *Oval, measuring about 32*16 µm.

*Usually contain a central undivided mass of protoplasm.

Taenia Segment


*Appears white and opaque and measures about 20 mm long

by 6 mm wide when freshly passed.

*Uterus has a central stem which has more than 13 main side

branches on each side.



*Appears gray-blue and translucent and measures about 13

mm long by 8 mm wide when freshly passed.

*Uterus has a central stem which has up to 13 main side

branches on each side.




*It is round to oval, measuring 33-43 µm.

*A thick, brown, radially striated wall surrounds the embryo.

*Hooklets (3 pairs) are present in the embryo.

H.nana Egg *It is colorless, oval or round, measuring 30-45 µm.

*Hooklets are present in the embryo.

*At the each end of egg thread-like structures called polar

filaments are usually visible.

D.latum Egg *It is pale yellow and oval in shape, measuring about 70*45


*It has an operculum.

*Contains a mass of granulated yolk cells surrounding an

undeveloped ovum.

*A small projection is sometimes visible at the non-

operculated end of the egg.



Egg *It is pale yellow-brown, large and oval, measuring about

140*85 µm.

*Contains an unsegmented ovum surrounded by many yolk


*Has a small operculum.

S.mansoni Egg *It is pale yellow-brown, large, and oval, measuring about

150*60 µm.

*Has a characteristic side spine.

*Contain a fully developed miracidium.

*A viable egg shows flickering of the excretory flame cells.

*A non-viable egg is dark-colored and shows no structural

detail or flame cell movement.

A.lumbricoides Fertilized


*It is yellow-brown and the shell is covered by an uneven

albuminous coat.

*Oval or round and measures 60*40 µm.

*Contains a central granular mass which is the unsegmented

fertilized ovum.

Unfertilized egg *It is darker in color and has a more granular albuminous


*More elongated, measuring about 90*45 µm.

*Contains a central mass of large refractile granules.

E.vermicularis Egg *It is colorless and has a clear shell.

*Oval in shape and usually flattened on one side. It measures

about 55*30 µm.

*Contains a larva.

S.stercoralis Larva *It is actively motile.

*It is large, measuring 200-300*15 µm and is unsheathed.

*Shows a typical rhabditiform bulbed esophagus.

*It can be distinguished from a hookworm larva by its

shorter buccal cavity.

T.trichiura Egg *It is yellow-brown and measures about 50*25 µm.

*Has a characteristic barrel shape with a colorless protruding

Mucoid plug at each end.

*Contains a central granular mass which is unsegmented




Egg *It is colorless with a thin shell which appears as a black line

around the ovum.

*Oval in shape, measuring about 65*40 µm.

*Contains an ovum, which usually appears segmented. If

specimen is more than 12 hours old, a larva may be seen

inside the egg. If the feces is more than 24 hours old than

larva may hatch and seen free in the feces.