Hookworm

 

Hookworm

Introduction

• Hookworm infection is a parasitic infection caused by the soil-transmitted helminths Necator americanus and Ancylostoma duodenale. Hookworms are blood-feeding nematode parasites that can cause mild diarrhea in both people and animals. Severe infection morbidity includes intestinal blood loss, anemia, and protein malnutrition.
  
• Zoonotic infection with hookworm species that do not use humans as a definitive host, such as Ancylostoma braziliense and Ancylostoma caninum (whose normal hosts are dogs and cats), can cause cutaneous larval migrans.

Geographical Distribution

• Hookworms are widespread in the tropics and subtropics. They are mostly found in areas with moist, warm climates where larvae can survive in the environment.
• N. americanus is the major cause of hookworm infection in the Americas, sub-Saharan Africa, and Asia.
• A. duodenale is found in more scattered, focal environments, specifically Europe and the Mediterranean.

Habitat

Adult hookworms live in the small intestine. They reside particularly in the jejunum The common site of infection for adult worms is the upper small intestine. Adult worms live in the lumen of the small intestine.

Morphology

• Adult worms:
• They look like an odd piece of thread and are about 1cm long. When living, they are white or light pinkish.
• The anterior part is curved, giving them the name hookworm. This bend forms a definitive hook shape.
• The head is slightly bent (hook). They have hooked mouthparts with which they attach to the wall of the gut, puncturing blood vessels and feeding on blood. The mouth carries characteristic teeth.
• Sex differences: The female is slightly larger than the male. Females are typically 12.5 mm, while males are 8mm. N. americanus males are usually 5-9 mm long, and females are about 1 cm long, making N. americanus generally smaller than A. duodenale.
• The male's posterior end is expanded in an umbrella-like fashion to form a copulatory bursa. This bursa is supported by fleshy rays. The pattern of distribution of these rays helps distinguish species. The cloaca is in the bursa, with two long, bristle-like spicules projecting from it.
• 
  Species differences in mouthparts: The mouth capsule of Ancylostoma duodenale shows the presence of four "teeth," two on each side. Necator americanus possesses a pair of cutting plates in the buccal capsule.
• Species differences in shape: A. duodenale has a single curve, looking like a 'C', while N. americanus has double curves, looking like an 'S'. The hook shape is much more defined in Necator than in Ancylostoma

                                    

• Eggs:
• Hookworm eggs are oval.
• They have a thin, colorless, hyaline shell. They are not bile-stained.
• They measure about 60–75 µm in length and 35–40 µm in width.
• Freshly passed eggs contain segmented blastomeres, typically 2-8 cells. When released in the intestine, they contain an unsegmented ovum, which develops during passage.

Life Cycle

• Eggs are passed in the stool.
• Under favorable conditions (moisture, warmth, shade), larvae hatch from eggs in 1 to 2 days.
• The released rhabditiform larvae (L1, the feeding non-infective stage) grow in the feces and/or soil, feeding on microbes.
• After 5 to 10 days and two molts, they become filariform (third-stage or L3) larvae that are infective. The L3 stage is non-feeding.
• On contact with the human host, these infective larvae penetrate the skin. This is the primary cutaneous route of transmission.
• (Note- Larvae can also infect orally by ingestion of L3 larvae present in the soil, although this occurs less frequently. Ingested larvae in the mouth develop directly into adult worms. A. duodenale can infect both through skin penetration and orally, while N. americanus larvae only infect through skin penetration.)
• Once they penetrate the skin, the larvae are carried through blood vessels to the heart and then to the lungs, and moulting occurs. They then penetrate the pulmonary alveoli, ascend the bronchial tree to the pharynx, and are swallowed.
• The larvae reach the small intestine, where they reside and mature into adults. The process from skin penetration to adult development takes about 5-9 weeks.
• Adult worms live in the lumen of the small intestine. In the small intestine, male and female worms mate.
• Female adult worms release eggs (approximately 9,000-10,000 eggs/day for N. americanus and 25,000-30,000 eggs/day for A. duodenale).
• Eggs appear in the stool and are passed in the feces.
• Most adult worms are eliminated in 1 to 2 years, but longevity may reach several years.

Pathogenesis

The pathogenesis and clinical symptoms depend on the site of the worms and their burden. Light infections may not be noticed.

• Pathogenicity of infective larvae:
• At the site of skin penetration, the infective filariform larva produces a local reaction called ground itch. This is a pruritic, erythematous, papular rash.
• Ground itch is also known as cutaneous larval migrans or creeping eruption. It is an allergic manifestation that can be more severe in Necator infection. Scratching can lead to secondary bacterial infections.
• For zoonotic hookworms (A. braziliense, A. caninum) that do not use humans as definitive hosts, the larvae migrate beneath the skin, which is the hallmark of cutaneous larva migrans. The movement beneath the skin leaves reddened, raised "tracts" or lines. As juveniles migrate, they cause serpiginous eruptions, a tingling sensation, and vesicles. Lesions are typically 3 mm wide but can expand to 15-20 cm. The larvae advance at a rate of a few mm per day and frequently change direction. The most common infection site is the feet. Zoonotic larvae usually die within weeks to months because humans are dead-end hosts.
• Migration of a large number of larvae through the lung produces minute hemorrhage and infiltration of leukocytes, potentially entrapping larvae in lung tissue.
• During the migratory phase in the lungs, minor cough and throat irritation may occur, though these symptoms are rare. Coughing, chest pain, wheezing, and sometimes fever can occur with large numbers of larvae in the lungs.
• Both eosinophilia (higher than normal level of eosinophils) and leukocytosis (high white blood cell count) occur at this stage.
• Pathogenicity of adult worm:
• The major pathological changes are caused by the attachment of adult worms to the intestinal walls by their buccal capsule. Attachment leads to mechanical disruption of the intestinal mucosa. The worm sucks in a portion of intestinal villi and utilizes gut epithelial cells and plasma for food.
• These parasites penetrate blood vessels with their mouth parts and suck blood for nutrition. These worms cause considerable loss of blood and tissue.
• Adult Ancylostoma can suck about 0.2 ml blood a day, while the smaller Necator suck about 0.03 ml per day.
• The worm's secretions contain anticoagulant activity, which adds to blood loss by causing continued bleeding from the attachment site. Excessive blood loss caused by heavy and prolonged infection leads to hypochromic microcytic anemia. Hypochromic means decreased red color, and microcytic means RBCs are smaller than usual.
• Major morbidity is caused by intestinal blood loss, iron deficiency anemia, and protein malnutrition, mainly resulting from adult worms ingesting blood, rupturing RBCs, and degrading hemoglobin.
• Pica may be caused by iron deficiency anemia.
• Early gastrointestinal symptoms include epigastric pain, indigestion, nausea, vomiting, and constipation. Diarrhea can occur early or later.
• Signs of advanced severe infection include those of anemia and protein deficiency, such as emaciation, cardiac failure, and abdominal distention with ascites.
• Light infections produce few or no symptoms but can include abdominal discomfort, diarrhea, and/or blood in the stool.

Laboratory Diagnosis

Microscopic Examination of stool- The most common method for diagnosing hookworm infection is microscopic identification of eggs in the stool. This is dependent on finding characteristic worm eggs. The recommended procedure for stool examination includes: collecting a specimen, fixing it in 10% formalin, concentrating it using the formalin–ethyl acetate sedimentation technique, and examining a wet mount of the sediment.

2.     Culture- Laboratory confirmation can also be made by microscopic identification of Ancylostoma or Necator species larvae cultured from the stool.

3.     Endoscopy- Identification of adult worms expelled after treatment or removed during endoscopy is also a method of laboratory confirmation. Adult worm identification may not be available at private laboratories.

4.     Imaging methods- can be used in the migratory phase of larvae in the lungs; a patchy infiltrate may be demonstrated on a chest X-ray.

5.     Blood tests are carried out to ascertain the nature of anemia (hypochromic microcytic anemia) and the presence of eosinophilia. Eosinophilia and leukocytosis occur during the larval migration stage.

6.     Occult blood test in the stool gives a positive reaction in case of hookworm infection. Charcot-Leyden crystals are often found in the stool in cases of hookworm infection. These are microscopic, needle-shaped structures made of galectin-10, a protein found in eosinophils, and are associated with eosinophil-rich inflammation and parasitic infections. They appear where eosinophil breakdown has occurred.

Treatment

• Mebendazole is a drug of choice, typically given orally at 100 mg twice daily for 3 days.
• Other antiparasitic drugs include pyrantel pamoate, thiabendazole, Albendazole, and levamisole. Albendazole and ivermectin can speed up recovery and alleviate symptoms of cutaneous larva migrans.
• Treatment of iron deficiency anemia with replacement iron therapy is necessary.

Prevention

• Sanitary disposal of human feces is essential. Avoid areas where human waste contamination of soil or water is likely. Do not defecate in places other than latrines/toilets. Do not use human excrement or raw sewage as fertilizer.
• Treatment of infected persons helps prevent contamination.
• Use of sanitary latrines and wearing footwear (shoes or other clothing) to prevent contact with soil are important measures. Do not walk barefoot in known infected areas.
• Good hygiene is important, including routine hand washing with soap and warm water, especially after touching animals or playing in soil. People may get hookworms by ingesting larvae from soil if they don't wash their hands.
• Thoroughly wash fruits and vegetables to remove soil/fertilizer residue.
• For animals, prompt removal of animal feces is the best prevention. Routine testing and deworming by a veterinarian are recommended for protecting animals. Deworming infected animals reduces environmental contamination and the chance of human exposure.

Preparation of bacterial smear

 

PREPARATION OF BACTERIAL SMEAR

Objective: To prepare a bacterial smear suitable for staining and microscopic examination, to observe bacterial morphology and arrangement

Theory:

A bacterial smear is a dried preparation of bacterial cells on a glass slide or it is a thin film of bacterial cells placed on a glass slide. This technique is a crucial step in the microscopic analysis of microorganisms, as it allows the staining and visualization of bacterial cells under different magnifications.

Smear preparation is especially important in simple and differential staining techniques such as Gram staining, acid-fast staining, or spore staining, which help in the identification and classification of bacteria. A well-prepared smear ensures that cells are not too thick (which may hinder observation) or too sparse (making it difficult to find bacteria under the microscope).

In our laboratory, the smear preparation process involves three essential steps:

1. Spreading of bacterial cells on the slide
2. Air drying the smear completely
3. Heat fixing the smear gently over a flame

Heat fixing kills the bacteria and adheres them to the slide. After this, staining is done to enhance visibility under a microscope, allowing the observation of bacterial shape (cocci, bacilli, spirilla), arrangement (chains, clusters, pairs), and basic internal structures.

Materials Required:

• Clean glass microscope slides
• Inoculating loop or sterile pipette
• Bacterial culture (broth or colony from solid media)
• Bunsen burner
• Staining reagents (e.g., crystal violet, Gram stain)
• Distilled water
• Bibulous paper or blotting paper
• Microscope

Procedure:

1. Take a clean grease free slide.
2. If using solid culture: Place a loopful of sterile water on the slide. Pick a small amount of bacterial colony using a sterile loop and mix it in the drop of water to create a uniform suspension.
   If using liquid culture: Take a loopful of culture and transfer it directly onto the slide.
3. Spread the suspension into a thin, even layer using the loop.
4. Allow the smear to air dry completely at room temperature. Do not heat to speed up drying, as it may distort the cells.
5. Once dry, heat fix the smear by quickly passing the slide 2–3 times through the flame of a Bunsen burner, smear side up. Do not overheat.
6. After fixing, apply the chosen stain (e.g., crystal violet for simple staining).
7. Allow the stain to sit on the smear for the recommended time (usually 1 minute).
8. Gently rinse the slide with distilled water to remove excess stain.
9. Carefully blot dry using bibulous paper or allow the slide to air dry.
10. Observe the slide under a compound microscope using 10x (to locate the smear), then switch to 40x and 100x oil immersion lenses for detailed examination.

Results:

• Draw the figure of the prepared bacterial smear as observed under the microscope.
  (Indicate cell shape and arrangement, e.g., cocci in chains, rod-shaped bacilli, etc.)
• During the smear preparation, the bacterial culture was successfully spread into a thin, even layer on the glass slide as shown in the figure.

Observation:

SN	Sample	Stain Used	Colour Observed	Shape	Inference / Result
1	E. coli	Safranin	Red	Rod-shaped (bacilli)	Red-colored bacilli observed

Conclusion:

The preparation of a bacterial smear is a fundamental and essential technique in microbiology. It allows the examination of morphological characteristics, such as shape, size, and arrangement, which are key in identifying and classifying bacterial species.

Precautions:

1. Always use clean, grease-free glass slides to avoid interference with the smear.
2. Use aseptic techniques while transferring bacterial cultures to prevent contamination.
3. Avoid overloading the slide with bacteria; a thin smear gives better results.
4. Allow the smear to air dry completely before heat fixing to avoid distortion of cells.
5. Do not overheat the slide during heat fixing, as it may damage the bacterial structure.
6. Handle the Bunsen burner and slides carefully to prevent burns and breakage.

References:

1. Cappuccino, J. G., & Welsh, C. (2017). Microbiology: A Laboratory Manual (11th ed.). Pearson Education.
2. Pelczar, M. J., Chan, E. C. S., & Krieg, N. R. (2001). Microbiology: Concepts and Applications. Tata McGraw-Hill.
3. Cheesbrough, M. (2006). District Laboratory Practice in Tropical Countries (Part 2). Cambridge University Press.

 

Ascariasis

 

Ascariasis

Introduction

Ascariasis is an infection of the small intestine caused by the nematode Ascaris lumbricoides. Ascaris lumbricoides is the largest intestinal nematode to infect humans. It is also known as the common roundworm. Historically, it was sometimes confused with the earthworm due to its resemblance. The specific name lumbricoides is derived from Lumbricus, the Latin word for earthworm.

Ascaris species are very large nematodes. Adult females measure 20 to 35 cm in length, and adult males measure 15 to 30 cm. In some sources, female length is noted as 20-40 cm and male length as 15-30 cm or 15-31 cm. While A. lumbricoides is the primary species involved in human infections globally, Ascaris derived from pigs (often referred to as A. suum) may also infect humans. These two are closely related, and hybrids have been identified, making their status as distinct species contentious.

Geographical Distribution

Ascariasis occurs worldwide. It is most common in tropical and subtropical areas where sanitation and hygiene are poor. The burden is highest in these regions. Main epidemic regions with a prevalence rate of approximately 10-90% include countries in South East Asia, Africa, and Latin America. The infection is generally rare to absent in developed countries. However, sporadic cases may occur in rural, impoverished regions of developed countries.

Habitat

The adult worm lives in the lumen of the small intestine. Specifically, 85% are found in the jejunum and 15% in the ileum. Humans are the only natural host and reservoir of infection. Natural infections with A. lumbricoides sometimes occur in monkeys and apes.

 Morphology

Adult Worm: Resembles an earthworm. It is elongated and cylindrical, tapering at both ends, with the anterior end being more pointed or slender than the posterior. The freshly excreted worms are yellowish-pink in color, gradually changing to white.The worm is sexually dimorphic.

Male Worm: Smaller than the female. Measures 15-30 cm or 15-31 cm in length and 2-4 mm or 3-4 mm in diameter or thickness. The posterior end is curved ventrally to form a hook and carries 2 copulatory spicules. The male tail has numerous genital papillae ventrally.

Female Worm: Larger than the male. Measures 20-35 cm or 20-40 cm in length and 2-6 mm or 4-6 mm in diameter or thickness. The posterior extremity is straight and conical.

Egg: Two types are liberated: fertilized and unfertilized.

• Fertilized Egg:
• Shape: Round to oval.
• Size: 50-75 µm x 40-50 µm or 60 x 40 µm.
• Covering: Surrounded by a thick smooth translucent shell consisting of three layers: the outer coarsely mamillated albuminoid coat (often stained brown by bile) (giving a rough surface), a thick transparent middle layer, and an inner lipoidal vitelline membrane. Some eggs found in feces may lack the outer mamillated coat and are called decorticated eggs.
• Bile Staining: Bile-stained, golden brown in saline mount.
• Floatation: Floats in saturated salt solution.
• Unfertilized Egg:
• Shape: Elongated and larger than fertile eggs (up to 90 µm in length). May be round to oval.
• Covering: Albuminous coat is thinner, distorted, and scanty. Bile Staining: Bile-stained, golden brown in saline mount.
• Floatation: Doesn’t float in saturated salt solution.

Life Cycle

• The life cycle of Ascaris completes in a single host, the human. There is no intermediate host.
• Stage I: Eggs in feces: Sexually mature females produce approximately 200,000 eggs per day which are passed with the feces in an unembryonated and non-infective form.
• Stage II: Development in soil: Embryonation occurs in soil given optimum conditions of warm temperature (20-25°C or 20°C to 30°C), sufficient moisture, and oxygen. The infective larva (L3) develops within the fertile egg in 18 days to several weeks or about 3-6 weeks or 10-40 days, during which the embryo moults twice.
• Stage III: Human infection and liberation of larvae: Humans get infected by ingesting embryonated eggs through contaminated food and water, especially fresh vegetables grown in fields manured with human feces ('night soil') or by direct transmission to the mouth through dirty fingers (fecal-oral route, geophagia) where soil contamination is heavy due to indiscriminate defecation. The inhaled eggs (via windswept dust) may also be swallowed.
• Stage IV: Migration of larvae through lungs: After infective eggs are swallowed, the larvae hatch in the duodenum, penetrate the intestinal mucosa, and are carried via the portal, then systemic circulation to the liver within 4-7 days of infection. They then travel via blood to the heart and to the lungs by pulmonary circulation. The larvae mature further in the lungs (10 to 14 days), where they moult twice and grow much bigger (from 0.2 mm to 2.0 mm). They break through the capillary wall and reach the lung alveoli.
• Stage V: Re-entry to stomach and small intestine: From the alveoli, the larvae ascend the bronchial tree to the throat and are swallowed, reaching the small intestine again.
• Development into adult worms: Upon reaching the small intestine, they develop into adult worms. They undergo two more moults, and sexual maturation occurs within 6-10 weeks.
• The female starts discharging eggs in the intestinal lumen, which are excreted along with feces, continuing the life cycle.

Pathogenesis

• There are two phases in ascariasis contributing to pathogenesis:
• Phase I: Migrating larvae:
• The migrating larvae cause pathological lesions.
• The severity depends on the sensitivity and nutritional status of the host and the number of migrating larvae.
• During migration and moulting through the lungs, larvae may cause pneumonia characterized by low-grade fever, cough, and other allergic symptoms, such as transient eosinophilic pneumonitis (Loeffler’s disease), elevated IgE, bronchospasm, dyspnea and wheezing, fever, and non-productive cough and chest pain.
• The sputum may be blood-tinged, and larvae may occasionally be found in it or more often in gastric washings.
• Phase II: Adult worm:
• Few worms in the intestine may produce no major symptoms or sometimes cause abdominal pain, especially in children. Most infections are asymptomatic.
• High worm burdens may cause abdominal pain and intestinal obstruction and potentially perforation in very high-intensity infections. Heavy worm burden in children can lead to intussusception and total obstruction.
• Large numbers of adult worms affect the nutritional status of the host, causing malnutrition, weight loss, diarrhea, and growth retardation in children. They can cause malabsorption of nutrition and protein-energy malnutrition and vitamin A deficiency.
• The metabolites of living or dead worms are toxic and immunogenic, producing allergic toxins that manifest as fever, conjunctivitis, and irritation. Hypersensitivity to worm antigens can also lead to urticaria, angioneurotic edema, and wheezing.
• Adult worms can produce trauma in host tissue.
• They may wander and block the appendical lumen or common bile duct and even the small intestine. They can enter the liver parenchyma, causing liver abscesses, go up the esophagus and out through the mouth or nose, or crawl into the trachea and lungs, causing respiratory obstruction or lung abscesses. Downward migration can cause obstructive appendicitis.

Sample Collection

• The stool specimen is the primary sample for diagnosing intestinal ascariasis.
• Preserve the stool specimen in formalin or another fixative.
• Sputum or gastric aspirate can be collected to identify larvae during the pulmonary migration phase.
• Adult worms are occasionally passed in the stool or through the mouth or nose.
• Bile obtained by duodenal aspirates may contain eggs.

Laboratory Diagnosis

• Microscopic identification of eggs in the stool is the most common method for diagnosing intestinal ascariasis. Both fertilized and unfertilized eggs may be present.
• The recommended procedure involves: collecting a stool specimen, preserving it, concentrating it using the formalin–ethyl acetate sedimentation technique, and examining a wet mount of the sediment.
• Where concentration procedures are unavailable, a direct wet mount examination of the specimen is adequate for detecting moderate to heavy infections.
• For quantitative assessments of infection, methods like Kato-Katz or quantitative fecal flotation can be used.
• If very few eggs are present, concentration techniques are crucial.
• Identification of larvae in sputum or gastric aspirate during the pulmonary migration phase. Fixed organisms should be examined for morphology.
• Identification of adult worms passed in stool or through the mouth or nose based on their macroscopic characteristics (e.g., presence of three “lips”).
• Serodiagnosis: Detection of Ascaris antibodies using tests like the Indirect hemagglutination test (IHA), Immunofluorescence assay (IFA), and Enzyme-linked immunosorbent assay (ELISA). Serodiagnosis is helpful in extraintestinal ascariasis, like Loeffler’s syndrome.
• Blood examination: A complete blood count may show peripheral eosinophilia, especially during the larval migration through the lungs.
• X-ray, ultrasonography, and CT scan can be used in diagnosis, particularly for detecting complications or adult worms. Chest X-ray may show patchy pulmonary infiltrates during larval migration.

Treatment

• Anti-parasite medications are the first-line treatment. Common medications include:
• Albendazole (single dose).
• Mebendazole (for 1-3 days).
• Ivermectin (single dose).
• Pyrantel pamoate (single dose). Safe for pregnant women.
• Piperazine citrate. Can be used via nasogastric tube for partial intestinal obstruction.
• These medications kill the adult worms. Side effects are usually mild, such as abdominal pain or diarrhea.
• Complete intestinal obstruction requires immediate surgical intervention.

Prevention

• Preventing fecal contamination of soil.
• Wash hands with soap and water before handling food, and teach children the importance of handwashing.
• 
Wash, peel, or cook all raw vegetables and fruits before eating, especially those grown in soil fertilized with manure or 'night soil'.
• Not defecating outdoors.
• Effective sewage disposal systems.
• Proper composting of manure to ensure the destruction of eggs.
• Treatment of vegetables and garden crops with water containing iodine (200 ppm for 15 minutes) can kill eggs and larvae.
• Improvement of personal hygiene.
• Treatment of infected persons.