Mechanism of Gram Staining & L forms

 

Mechanism of Gram Staining

• Christian Gram - Gram stain in 1884- based on the differences in cellwall

• The difference between gram-positive and gram-negative bacteria is due to the physical nature of their cell walls. If the cell wall is removed from gram- positive bacteria, they become gram negative. 

                            

• During the procedure the bacteria are first stained with crystal violet and next treated with Gram’s iodine (mordant) to promote dye retention. 

• When gram-positive bacteria then are decolorized with ethanol, the alcohol shrinks the pores of the thick peptidoglycan. Thus the dye-iodine (CV-I) complex is retained during the short decolorization step and the bacteria remain purple. 

• In contrast, gram-negative peptidoglycan is very thin, not as highly cross-linked, and has larger pores. Alcohol treatment also dissolves lipid from the gram- negative wall to increase its porosity further. For these reasons, alcohol more readily removes the purple crystal violet-iodine complex from gram-negative bacteria. They then absorb the colour of counter stain – Safranin. 
  

• Thus, Gram-positive bacteria stain purple, whereas gram-negative bacteria colour pink or red 

  

Effect of Lysozyme, Penicillin on Cellwall

The enzyme lysozyme attacks peptidoglycan by hydrolysing the bond between N-acetylmuramic acid with N-acetylglucosamine

• Penicillin inhibits peptidoglycan synthesis; inhibits transpeptidases which perform crosslinking of peptidoglycan . 

In an osmotically protective medium, Gram-positive bacteria are converted to protoplasts which completely lack a wall.  Gram-negative cells retain their outer membrane after penicillin treatment and are classified as spheroplasts because some of their cell wall remains.

• Protoplasts and spheroplasts are osmotically sensitive - in a dilute solution, they will lyse due to uncontrolled water influx

• Mycoplasmas lack a cell wall (pleomorphic) and are osmotically sensitive, can grow in dilute media or terrestrial environments because of stronger plasma membranes; the presence of sterols in the membranes provide added strength.

 Such protoplasts and spheroplasts are non-viable and cannot multiply, If they grow and divide, they form L forms

L-phase bacteria, L-phase variants, and cell wall-deficient (CWD) bacteria

• bacteria that lack cell walls

• When bacteria are treated with enzymes that hydrolyze the cell wall (e.g. lysozyme) or antibiotics that interfere with biosynthesis of peptidoglycan (penicillin), wall-less bacteria are often produced. 

• Such a treatment of bacteria in osmotically protective medium liberates protoplasts from gram positive bacteria and spheroplasts from gram negative bacteria. Spheroplasts retain the outer membrane.  

• Usually these treatments generate wall-less non-viable organisms that do not multiply. However, if such cells can grow and divide, they are called L forms.

• L forms were first reported by Klieneberger Nobel in cultures of Streptobacillus monoliformis. They are named L forms after Lister Institute, where they were discovered. 

• They are produced more readily with penicillin than with lysozyme.

• Some L forms are stable and some are unstable.  

• Unstable forms (spheroplasts) are those which revert back to cell wall containing state when inducing stimulus (penicillin) is removed. Such forms usually have small amounts of residual peptidoglycan that serves as primer for building cell wall. 

• Stable forms ( protoplasts) do not revert back to normal form since they completely lack peptidoglycan. 

Significance of L forms: L forms may produce chronic infections in the host. They may persist in protective regions of the body. Since L forms are relatively resistant to antibiotics, they are difficult to treat. Their reversion to normal form can result in relapse of infection.

• Since they lack cell wall, they don’t have a definite shape. 

• L forms are difficult to cultivate and require medium that has right osmotic strength and low concentration of agar, inactivated serum and sucrose. 
• L forms resemble mycoplasma in morphology, type of growth on agar “fried-egg colony”.

• Mycoplasma, also lack a cell wall but not considered L-forms since they are not derived from bacteria that normally have cell walls. While mycoplasma lack cell wall and have sterols in their membrane, the L forms may have reminiscent of cell wall but do not have sterols in their membrane.

 

Hepatitis B - Laboratory diagnosis & Prophylaxis

 Laboratory diagnosis

 
HBsAg first appears in the blood during the incubation period, while the virus is actively 'replicating' in liver cells. The antigen is produced in vast excess - it is associated with new infectious virus particles as well as occurs in the serum as small non-infectious spherical and filamentous forms.

In acute infection, the HBsAg usually disappears within 3 months of onset. 

The HBeAg and HBV DNA can also be detected in blood while the virus is actively replicating in the liver . Symptoms usually appear as the concentrations of bilirubin, alanine aminotransferase and each of the major viral components peak in the serum. These events coincide with the first appearance of antibodies to HBV proteins

Antibody to HBcAg (anti-HBc) rises first. As a generalisation, the detection of IgM antibody specific to the hepatitis B core is the primary indicator of acute infection. It usually appears at or just before the onset of symptoms and remains detectable for at least 6 months. The IgG component of anti-HBc usually persists for life. 

Anti-HBe is the second antibody to appear and is associated with the rapid clearance of HBeAg. Later, anti-HBe declines and persists for only a few months or years if there is no active viral replication.

The antibody to HBsAg, anti-HBs, may not become detectable for 3-6 months after acute infection. It is associated with resolution of the illness. This antibody is recognised as the marker of immunity to HBV.

Detection of HBsAg + IgM HBcAg (anti-HBc) -recent/acute infection

Detection of HBsAg + IgG HBcAg (anti-HBc) -remote infection

Detection of HbeAg - High infectivity

Nondetection of HbeAg-low infectivity

Primary & secondary tests to diagnose/monitor hepatitis B virus (HBV) infection

	Marker	Incubation period	Acute infection	Past/resolved infection	Chronic infection	Vaccination
For diagnosis and monitoring
	HBsAg	±	+	–	+	–*
	Anti-HBs	–	–	+	–	+
	Anti-HBc- Total	–	±	+	+	–
	Anti-HBc- IgM	–	+	–	±†	–
	HBeAg	±	+	–	±	–
	Anti-HBe	–	–	±	± ‡	–
	HBV-DNA	±	+	±	+	–

^*Recent HBV vaccination within one to two weeks can lead to a false-positive test. The vaccine antigen can be detected at low levels;

^†May be positive in chronically infected individuals;

^‡Patients with chronic HBV infection usually have detectable Hepatitis B e antigen (HBeAg) or antibody to hepatitis B e protein (anti-HBe). Rarely, both HBeAg and anti-HBe can be detected simultaneously;

Interpretation of HBV Immunologic Markers

Markers				Interpretation
HBsAg*	HBcAb†	HBsAb‡
–	–	–	HBV infection
–	–	+	Immune because of vaccination
–	+	+	Immune because of natural HBV infection
+	+	–	Acute or chronic HBV infection
–	+	–	Interpretation unclear; four possibilities: l  Resolved HBV infection (most common) l  False-positive HBcAb l  “Low-level” chronic HBV infection l  Resolving acute HBV infection

---

HBcAb = hepatitis B core antibody; HBsAb = hepatitis B surface antibody; HBsAg = hepatitis B surface antigen; HBV = hepatitis B virus; + = positive test result; – = negative test result.

*— The presence of HBsAg indicates that the person is infectious.

†—HBcAb appears at the onset of acute HBV infection. Presence may also indicate chronic HBV infection or a false-positive test.

‡—The presence of HBsAb indicates recovery and immunity from HBV infection or successful immunization against HBV.

A recently infected chronic carrier will show evidence of ongoing viral replication in liver cells. HBeAg and HBV DNA can be detected in blood for months or years after acute infection.

Treatment

No specific antiviral treatment  for acute HBV infection. The main treatment is symptom relief.  Special consideration should be given to any medications that a patient takes to see the effect it may have on an impaired liver.

Interferon alpha alone or in combination with other antiviral agents such as lamivudine or famciclovir is useful in treating chronic hepatitis.  There is no treatment for carrier state though some show spontaneous resolution. 

Patients should avoid alcohol and cigarettes which can aggravate the liver.  

The only safe and effective measure for prevention is universal active immunisation. In 1992, WHO has recommended the integration of Hepatitis B vaccine into the immunisation programmes of all nations. India is lagging behind due to the high cost of imported vaccine. Development of low cost vaccine in India should help include this in national immunisation schedule.

Hepatitis B

Hepatitis B is a viral infection that attacks the liver and can cause both acute and chronic disease.

Hepatitis B virus- DNA virus belonging to the Hepadnaviridae family 

1965, Blumberg-discovered Hepatitis B surface antigen from the serum of an Australian aborigine, hence named Australian antigen

Family: Hepadnaviridae

Genus: Orthohepadnavirus

Species: Hepatitis B virus

Structure  

Contains icosahedral nucleocapsid, the core (27 nm) and outer envelope (42 nm)

There is an outer shell (or envelope) composed of lipid and protein that is termed “surface antigen” or “HBsAg”.

Inner protein shell that is referred to as the core particle or “HBcAg”, contains the viral DNA and enzymes used in viral replication ( “DNA polymerase”).

HBeAg (hepatitis B e antigen) is also associated with the nucleocapsid of HBV.  It is soluble, secreted and seen in circulation

 

Three types of viral particles are visualized in infectious serum by electron microscopy.

• 42 nm in diameter (Dane particle- Complete Virion)
• 20 nm in diameter (Spherical Structures)
• 22 nm in diameter (Filamentous Particles)

 The 42-nm spherical Dane particle (Associated with it is, HbsAg) can be disrupted by nonionic detergents to release the 27-nm core that contains the partially double-stranded viral DNA genome. Associated with the core is, HBcAg, hepatitis B core antigen. A soluble antigen, termed hepatitis B e antigen (HBeAg), may be released from core particles by treatment with strong detergent. .

Genome 

• Is partially double-stranded DNA held in circular configuration 
• One strand incomplete (positive), associated with it is a viral DNA polymerase with DNA dependent DNA polymerase and RNA dependent reverse transcriptase activity

• The genome is 3020–3320 nucleotides long (for the full length strand) and 1700–2800 nucleotides long (for the short length strand).

• Compact organization, with four overlapping genes (S, C, P, X genes) and no non-coding regions.
• S gene, codes for Surface antigen HbsAg 
• C gene, codes for nucleocapsid Core antigen- HbcAg and a soluble antigen-HbeAg 
• HbcAg is associated with the nucleocapsid core and is not secreted or circulated in blood -can be detected in hepatocytes
• HbeAg is secreted and present in circulation during infection-presence indicates HBV replication and infectivity
• Detection of HbeAg -marker for HBV 
• P gene, for DNA polymerase
• X gene, for nonparticulate antigen, HbxAg-has activating effects on viral and some cellular genes.
• HbxAg- present in patients with severe chronic hepatitis and hepatocellular carcinoma

General symptoms include

· Fever

· Nausea and vomiting

· Loss of appetite

· Flu-like symptoms

· Fatigue and weakness

· Lack of nutrition

· Aching in the abdomen

· Pale or clay-colored stool

· Dark urine

· Yellowing of skin or eyes

Ø  Symptoms similar to HAV; but severe and prolonged; Onset is insidious, fever not prominent

Ø  Extrahepatic complications – arthralgia, urticaria,  polyarteritis, glomerulonephritis (due to circulating immune complexes carrying the viral antigens)

Ø  90-95% adults with acute Hep B infection recover in 1-2 months- remain immune thereafter

Ø  0.5-2% cases- mortality, more in post transfusion cases

Ø  1% adults, with simultaneous delta virus infection develop fatal fulminant hepatitis

Ø  1-10% cases, remain chronically infected- asymptomatic carriers or develop recurrent/chronic liver disease or cirrhosis- some develop hepatocellular carcinoma

Ø  Vaccine against HBV present- hepatocellular carcinoma is the only human cancer which is vaccine preventable

 Pathogenesis is immune mediated

Ø  Hepatocytes carry viral antigens- Attacked by antibody dependent NK cells and cytotoxic T cells

Ø  In the absence of adequate immune response, HBV infection may not cause hepatitis but lead to carrier state

Ø  Infants and immunodeficient persons become asymptomatic carriers following infection

Epidemiology

•        Hepatitis B occurs throughout the world- no seasonal distribution

•        Natural Infection only in humans

•        Virus maintained in carriers- their blood contains circulating viruses for a long time, even lifelong

•        Infection is usually sporadic- Occasional outbreaks occurs mostly in hospitals, orphanages and institutions for mentally handicapped.

•        The prevalence of hepatitis carriers varies- Overpopulated, underdeveloped regions have high endemicity , developed countries have low endemicity

•        India has intermediate endemicity (2-7% carriers), with higher rates in the southern part of the country

 

Carriers

Super carriers

•    Highly infectious

•    High titre of HBsAg along with HBeAg, DNA polymerase and HBV in circulation

•     Elevated transaminases

•        Enormous antigenemia and viremia (10¹³ HBsAg particles and 10⁸ HBV per ml blood)

Simple Carriers

• Low infectivity
• Low titre HBsAg in blood, with negative HBeAg, DNA polymerase and HBV in circulation
• Elevated transaminases

Transmission

HBV is a bloodborne virus and the infection is transmitted by parenteral, sexual and perinatal modes.

Blood of carriers, and less often of patients, is the most important source of iniection. The virus may also be present in other body fluids and excretions, such as saliva, breast milk, semen, vaginal secretions, urine, bile and faeces. Of these, semen and saliva are known to transmit the infection; others may also do though much less efficiently than blood. Faeces are not known to be infectious.

Transfusion of carrier blood, once the most widely known mode of infection has largely been eliminated wherever donor screening is strictly enforced. Therapeutic and prophylactic preparations from pooled human blood and serum have led to hepatitis but this risk is now minimal, with sçreening of donors and production techniques ensuring virus inactivation. However, HBSAg screening is not a totally safe method as infection has occurred even with HBSAg- and anti-HBc positive blood, which may have had undetectable amounts of virus.

Many other therapeutic, diagnostic. prophylactic and even nonmedical procedures are now the main modes of infection.  HBV is very highly infectious, far more than HIV. Any object or procedure than can convey minute traces of infected blood or other material,  can be infectious.

Infection by direct contact with open skin lesions such as pyoderma, eczema, cuts and scratches is very common among young children in developing countries, as also through household transmission where opportunities exist for contact with blood or saliva among members.

HBV has been said to survive in mosquitoes and bed bugs for about 2 weeks after blood meal, but no virus multiplication occurs. They do not appear to transmit the infection.

Congenital or vertical transmission is quite common from carrier mothers. The risk to babies is high if the mother is HBeAg positive (60-90 per cent) and low if negative (5-15 percent). True congenital infection (in utero), transplacental is rare. Infection is usually acquired during birth by contact of maternal blood with the skin and mucosa of the fetus, or in the immediate postnatal period.

Infection by ingestion has been reported, but its efficiency is very low. It is better that carrier mothers do not breastfeed when proper nutrition of their babies can be otherwise ensured. HBV infected neonates generally do not suffer from any clinical illness but remain carriers tor life and some of them may develop hepatocellular carcinoma after many decades

Sexual transmission of HBV occurs everywhere, but is more important in the developed countries particularly in the promiscuous homosexual. The risk of transmission by heterosexual and homosexual contact increases with the number of partners and the duration of Such relationships. HBV infection has occurred after artificial insemination. Semen donor screening is therefore obligatory.

Certain groups and occupations carry a high risk of infection. These include medical and paramedical personnel, staff of blood banks, dialysis units, medical laboratories and mental health institutions, barbers and sex workers. Dentists and doctors have been responsible for small outbreaks. In countries like Britain, HBV carriers are barred from invasive medical practice. Carriers are also not permitted to be medical Students.