The Complications of Surgical Treatment of Grade IVFilarial Lower Limb Lymphedema

Breast filariasis or inflammatory breast carcinoma? Reaching a diagnosis.

Recent advances in the Diagnosis and treatment of Lymphatic Filariasis.

Recent advances in the Diagnosis and treatment of Lymphatic Filariasis.

                                             Dr. Dwijesh Kumar Panda, M.D, Ph.D(Medicine-Filaria)

Introduction;

• Caused by nematodes those  inhabit the lymphatics and subcutaneous tissues.
• Species are Wuchereria bancrofti  (95%), Brugia malayi, and Brugia timori.
• It is usually acquired in early childhood.
• Major cause of disfigurement and disability.
• More than 120 million people worldwide are affected.
• India accounts for 40% of global prevalence. (Map)
• Transmitted by mosquito vectors. Humans are definitive host. (Fig 1-Life cycle)
• Adult worms live for 5-7 years.
• More than $ 840 million is lost each year.

Lymphatic Pathology:  In chronic lymphedema, lymph flow is stifled. Macromolecular protein deposition increases. Fibroblasts accumulate in interstitium. Fibrovascular proliferation results in worsening brawny edema. Obliteration of elastic fibers. Increased collagen deposition and fibrosis. Fig.

Clinical presentations:

Diagnosis:

• Definite diagnosis by (a) detecting circulating filarial antigen. Og4C3 test procedure chart.
• Demonstration of microfilariae in the blood- rarely seen after an attack of lymphangitis due death of adult worm. Picture

• Filarial DNA in the blood. (PCR) Polymerase chain reaction used as a research tool to detect filarial infection in humans. Not commercially available. One pg of W. bancrofti genomic DNA added to 100 microliters of human blood.
• Adult worms in the lymphatics- rarely detected.
• Peripheral blood Eosinophilia- may exceed 3000/micro L. Other helminth infections frequently coexist- may increase eosinophils in the blood.
• Tropical Eosinophilia- Confused with Tuberculosis, Asthma, Round worm infestation. Leads to progressive interstitial fibrosis, Hypersensitive immunological process. Total eosinophil count more than 3000/micro L.  Fig

Nonspecific diagnosis: Elevated serum IgE. Microscopic hematuria, Proteinuria and chyluria. Picture.

• OG4C3 Test:   .

Detection of circulating adult filarial antigen in the blood. Positive even in amicrofilaremic          individuala.

The assay has excellent specificity and better sensitivity than microscopy.

The filarial antigens become undetectable in treated or “burned out” infection.

The quantitative OG4C3 test is useful in following patients after treatment as antigen levels decline with treatment.

Imaging:

• Ultrasound techniques used to detect presence of adult worms in lymphatic vessels.
• Living worms in continuous motion is described as “filarial dance” sign. Movie
• Worms can be visualized in breast and scrotal lymphatic.
• Lymphoscintigraphic technique:  Useful for assessing the extent of lymphatic damage in the regional lymphatic prior to development of clinical symptoms. Massive lymphatic dilation observed surrounding adult worms. The images cannot provide high resolution anatomical details. A small amount of radio isotope labeled colloidal substance is injected intra-dermally.

Treatment:

• Medical:

• Treatment of choice- Diethyl carbamazine (DEC) – Dose: 6mg/kg for 12 days alone or in combination with Albendazole- 400mg-once.
• Ivermectin- It has microfilaricidal activity. No precedence over DEC in Bancroftian filariasis.
• Benzopyrones and Flavonoids improve lymphedema through protein resorption and proteolysis.
• Diuretics have no effect.
• Antibiotics reserved for bouts of cellulitis.
• Asymptomatic phase:  Due to the Cytokine IL-4 suppressing the activity of TH1 cells of immune system. This occurs for years until inflammatory reaction rises again.
• Restriction of long chain triglycerides have some benefit.

• Surgical:        

• Largely disappointing.  
• MLVA- Microlymphatic-venous anastomosis- encouraging in early stages of lymphedema.
• Overall result disappointing in long-term edema.

                                                    THANK YOU.

Homage to Dr(Prof) L.N.Mohapatra, my Ph. D guide.

Wolbachia and filarial febrile episode

Wolbachia and filarial febrile episode:

Lymphatic filarial parasites contain endosymbiotic bacteria, Wolbachia.

These are rickettsia-like organisms required for homeostasis of the host parasite.

The inflammatory manifestations of lymphatic filariasis are attributable to immune responses directed to Wolbachia.

Filarial parasites stimulate innate inflammatory responses.

This is absent if the bacteria have been cleared with antibiotics.

Severe inflammatory responses have been observed when Wolbachia are released into the blood following death of the worms after antifilarial chemotherapy.

The pathogenic role of Wolbachia endosymbiotic bacteria in filarial diseases  is proved. The early inflammatory responses including TLR4-mediated signalling, chemokine and cytokine responses and inflammatory cell recruitment is seen in filarial disease.  The impact of the discovery and characterization of Wolbachia on filarial  worm is ascertained.

Wolbachia bacteria in a section of female Wuchereria worm.

Og4C3 ELISA TEST for Filaria

Og4C3 ELISA TEST for Filaria

This test is used to diagnose infection with wuchereria bancrofti The diagnosis is based on the use of a monoclonal Ab Og4C3 which is coated on to the surface of microtitre plates. The coating monoclonal antibody (Og4C3) has been shown to specifically recognise only Wuchereria bancrofti antigen in human sera. Principle:

1.Microtitre Plates Five U-bottom polystyrene microtitre plates pre-coated with Og4C3 monoclonal antibody. 2.Solution A (green solution) One 50 ml bottle for primary treatment of serum samples. 3.Solution B (YELLOW solution)One 50 ml bottle for secondary treatment of serum samples. 4.Antibody and conjugate diluent One 100 ml bottle (blue soloution) 5.Standard antigens (1-7) Seven dilutions of Onchocerca gibsoni

antigen. Each vial contains 600 ml of standard (orange cap) 6. Rabbit and-onchocerca antibody One 300 ml bottle(yelow cap) 7.Anti-Rabbit conjugate One 300 ml bottle (purple cap.) ' 8.Chromogen One 60ml bottle of single component ABTS ready to use. 9.Washing buffer(X20) concentrate.

METHOD

All steps carried out at room temperature . Ensure that all reagents and the microtitre plates are at room temperature before use. Quantities indicated below refer to those required for the use of ONE plate. To prepare washing buffer, dispense one measure from the x20 dispensing bottle into 500 ml of distilled water. 1. Preparation of serum samples. Add 50 ul of each serum sample to 50 ul of solution A (green solution) in a suitable container.(e.g. any type of 96- well microtitre plate, BUT NOT one of the coated plates supplied with this kit.) Mix the contents of the wells by tapping the plate, and leave at room temperature for 2 minutes. Using a multichannel pipette ,add 50ul of solution B (YELLOW solution) to each well containing the serum sample/solution A mixture. Mix well by pipetting u p and down several times, and IMMEDIATELY transfer 50 ul of the treated serum to each of two wells of the og4C3 Ab-coated plates supplied. Up to 40 samples in duplicate can be tested per plate (see the plate layout diagram)

Add 50 ul of each Standard Antigen (no1- 7, Orange caps ) directly to duplicate wells of columns 11 and 12.Add the standards directly to the wells; DO NOT treat or dilute in any way. For conjugate control (cc), add 50 ul of solution B to wells A11 and A12. 3. Place the plate in a humid container and incubate for 1.5 hours. 4. Wash the plate three times with wash buffer, invert and tap gently to remove residual droplets. 5. Prepare rabbit anti-Onchocerca antibody by adding 50 ul of rabbit anti-onchocerca antibody (YELLOE cap) to 6ml of antibody diluent (Blue solution) Add 50 ul of the diluted rabbit antibody to all wells and incubate for one hour 8. Wash the plate three times as before. 9. Add 100 Ul of chromogen (ABTS)(do not dilute) to each well and incubate for one hour. 10.Plates can be read with a spectrophotometer at a single wavelength of 414 and 492 nm. Blank the plate reader on wells containing conjugate control or a row of wells containing substrate in a separate blanking plate.

INTERPRETATION OF RESULTS

A standard curve may be drawn using the optical densities of the seven standard control samples as the Y values.

The X values can be the numbers 1 to 7 corresponding to the controls. The test samples can be allocated to groups on the basis of the optical density compared with the optical density of the standard curve samples. Samples with an OD>= Standard No 2 are considered to be the positive samples.

BACK

| Background | Objectives |Progress | Related links | Contact us |

Send your queries to vcrc@vsnl.com

Designed and maintained by VCRC
copy right © vcrc 2000-2006

Bancroftian filariasis causes Male Sterility

Bancroftian filariasis causes Male Sterility.

Dr Dwijesh Kumar Panda.

Lymphatic filariasis is caused by Wuchereria bancrofti. It constitutes about 99.4% of all infections. It is the commonest cause of lymphoedema and hydrocele in India. It is the second most common cause of physical disability next to malaria among the debilitating vector- borne tropical diseases (WHO). Among the endemic countries, the highest incidence of LF is in India, which harbours 40 % of the world disease burden (Michael et al., 1996). It is reported that 40.65 million episodes of acute adenolymphangitis (ADL

)occurs annually in the affected population in India. It is estimated to cause a wage loss of US dollar 811 million annually (Ramaiah et al.,2000).The acute ADL attacks cause genito-urinary problems such as hydrocele, lymphoedema of the genital region or chylocele associated with sexual dysfunction (Dreyer et al, 1997).It is well known that the initial damage to the lymph vessels is caused by the adult filarial worms. .The acute ADL attacks and the consequent progression of the disease are caused mostly by bacterial infections in the affected parts.The damaged lymphatics promote development of lymphoedema involving genitalia. It is the rupture of damaged lymph vessels that cause hydrocele, chylocele, chyluria, lymph scrotum or lymphorrhoea. Death of adult worms in the vicinity of testes is known to result in acute onset of of hydrocele and funiculitis. Filarial worms live in the lymphatic and lymph nodes causing dilation and incompetence of their valves resulting in lymph stasis and oedema. This can affect hydrocele and genitals

more commonly than lower limbs (80-85%). The plasma proteins in the interstitial fluid cannot pass through venous capillaries due to their high molecular weight. These are cleared through lymphatics which have larger caliber.  Inadequate clearance of proteins by lymphatics causes high protein oedema called lymphoedema. By the time it is clinically manifest, the damage to the lymphatics is already advanced and the lymphatic structure  is not reversible.So lymphedema is not curable by available methods of treatment.

  

Wolbachia species are rickettsia-like bacteria found intracellularly within filarial nematodes of Wuchereria.Wolbachia release endotoxin like  molecules that are thought to play a role in the pathogenesis of Wuchereria infections. Evidence for this includes the use of doxycycline, which kills the Wolbachia resulting in a reduction of the inflammatory response to the nematode infection.

Cytokines influence the nature of the immune response. These are small molecules that signal between cells inducing regulation of immunity. They are referred to as interleukins (IL) if produced primarily by leukocytes. IL-1 produced by macrophages and epithelial cells activates vascular endothelium, tissue destruction, fever, mobilization of polymorphonuclear leukocytes and induction of acute phase proteins(CRP). IL-4 produced by mast cells activates IgE response (Allergy). In lymphatic filariasis, the stagnation of lymph promotes bacterial growth which results in acute ADL attacks, observed frequently in the affected limbs and genital region. Many recent studies have demonstrated the role of microorganisms, mostly streptococci to be causative organisms for ADL attacks(Suma et al,1997; Vincent et al.,1998). The liberation of intermittent interleukins by the inflamed and infected tissues cause episodic fever, pain and fibrosis. So in repeated ADL attacks the long term antibiotic therapy, long acting parenteral benzathine penicillin is indicated.(Goodman & Gilman.)

Case studyt:-1.

Parents brought a young man of 25 years complaining of pain in the right testis and right inguinal area since several months. The pain is referred to the right side of lower abdomen. The size of the right testis is smaller in comparison to the left. Few years back both sides were normal. The size is gradually decreasing. There is a swelling on the middle of the rt inguinal ligament and slightly above. The photograph and ultrasound picture is enclosed for reference. The person is married since 2 years without any issue. No contraceptive measures taken.

The ultrasound reveals that the right testis is small and rudimentary. No color flow could be appreciated. Rt. Epididymis and right cord also appears thinner. Left testis, Epididymis and cord appears normal except the head and tail appears mildly swollen.

The seminal fluid analysis reveals oligospermia. The sperm count is 16.2 million/ml. The viscosity is low.The circulating adult filarial antigen test (Og4C3) is positive (512 Antigen Unit)

Case study-2.

A person aged about 33 years is married since more than 5 years without having any issue. His testis of both sides are gradually shrinking. He complains of recurrent pain and swelling in the inguinal region. He has lost sex drive. He is getting fever from time to time along with swelling and tenderness in the scrotum. The ultrasound report and photograph reveals swelling of both epididymitis. The outer area appears irregularly echogenic due to fibrosis. Right cord appears swollen and measures 8.75 mms, normal being below 5 mms. The diagnosis is right Funiculitis with bilateral chronic Epididymitis. Seminal fluid analysis was done. The total sperm count was 9.4 million/ml, low viscosity with non-motile dead spermatozoa about 40%. The circulating adult filarial antigen test (Og4C3) is positive (512 Antigen Units)

Og4C3 Test;- It is a highly sensitive and specific quantitative monoclonal antibody based ELISA test for the diagnosis of filariasis.

Discussion;-

The clinical findings, laboratory diagnosis and ultrasound report and photograph confirms the diagnosis of Bancroftian filariasis. Repeated inflammation of the epididymis and the testis caused atrophy of the male reproductive organ. Inflammation raised the temperature if the testis. The optimum temperature of the testis should be below 3 degree centigrade of normal body temperature of  37 degree centigrade. More than 250 million sperm are produced daily inside coiled tubes called seminiferous tubules. Immature sperm are moved to the epididymis, where they mature and start to swim. Mature sperm are stored both in the epididymis and in the first section of the vas deferens. Constant high temperature in the testis by course of inflammation caused atrophy of the testis leading to loss of spermatozoa. Bancroftian filariasis is an important cause of inflammation of the testis, epididymis and vas deferens. It causes male sterility in an endemic zone of filariasis.

Nectar for Filaria from the tree of sorrow

Nectar for Filaria from the tree of sorrow

Ganga Shiuli (ଗଙ୍ଗ ଶିଉଳି) Nyctanthes abor- tristis Linn

Dr. Dwijesh Kumar Panda

M. D., Ph. D. (Medicine)

Abstract

Nyctanthes abor-tristis Linn is the one of the well-known medicinal plant. It is a common wild hardy large shrub or small tree. It is endemic of India, distributed wild throughout the country. Different parts of this plant are used in Indian systems of medicine for various pharmacological actions.

Introduction

Nyctanthes abor-tristis Linn is a small sacred ornamental tree known across the country for its fragrant white flowers. It is commonly known as Night Jasmine or Parijata.

Sanskrit: Parijatha  

Hindi: Harsingar

Oriya: Gangasiuli

Taxonomic classification

Kingdom: Plantae

Family: Oleaceae

Genus: Nyctanthes

Species: arbor-tristris

Botanical description:

Nyctanthes abor-tristis Linn is a large shrub growing to10m tall; with flaky grey bark and rough leaves. The flowers are fragrant, with a five to eight lobed white corolla with an orange red center, opening at dusk and finishing at dawn. The leaves are opposite, simple, 6-12 cm long and 2-6 cm broad.

Traditional uses:

The flowers are collected for religious offering and to make garlands. The orange heart is used for dyeing silk and cotton, a practice that started with Buddhist monks whose orange robes were given their color by this flower. The Parijata is regarded in Hindu mythology as one of the five wish granting trees of Devaloka.

Different parts of Nyctanthes abor-tristis Linn are known to possess various ailments by tribal people of India esp. Odisha and Bihar along with its use in Ayurveda, Sidha and Unani systems of medicines. It is used in several ailments including sciatica, rheumatism, gout and other joint diseases.

Flower:

The flowers are used as stomachic, carminative, astringent to bowel, antibilious, expectorant, hair tonic and in the treatment of piles and various skin diseases. The bright orange corolla tubes of the flowers contain a coloring substance nyctanthin, which is identical with α- Crocetin (C20H24O4) from Saffron. The corolla tubes were formerly used for dyeing silk, sometimes together with Safflower or turmeric.

The indigenous people of Chittoor district, Andhra Pradesh (India) widely use the whole plant for treatment of cancer, root for fever, sciatica, anorexia; bark as expectorant.

Chemical constituents:

Recent researches reported the isolation of polysaccharides, iridoid glycosides, B-sitosterol, B-amyrin, glycosides, nyctanthoside-a, iridoid, nyctanhic acid, oleanolic acid, arbortristosides- A,B,C  have been isolated from this  plant. Khatune et al., in 2003 isolated  4, hydroxyhexahydrobenzofuran -7- one from chloroform extract of flowers of Nyctanthes arbor-tristis. It was formed to be antibacterial, larvicidal.

                   

4 hydroxyhexahydrobenzofuran-7-one Arbortristoside-A Arbortristoside-B

Flowers contain essential oils, coloring matter (nyctanthin), mannitol, tannin and glucose.

Reported pharmacological profile:

Ethanolic extract of fresh flowers of Nyctanthes arbor-tristis Linn was tested for its antispasmodic activity using guinea pig ileum. It was found to inhibit contractile response of acetylcholine. The flowers ethanolic extract was tested for its antihelmintic activity using  piperazine citrate as a standard. The antihelmintic activity was studied on the basis of inhibition of contractile effect of acetylcholine by various dilutions of this extract. Khature et al showed that this extract is not carcinogenic as it inhibits EAC cell growth. This compound possesses no adverse effect on central nervous system. It was found to significantly inhibit acute inflammatory edema produced by histamine, 5-hydroxytryptamine and hyalouronidase in hind paw of rats. It also reduced acute inflammatory swelling in the knee joint induced by turpentine oil. The anti-inflammatory activity of arbortristoside-A may be due to the inhibition of the action of prostaglandin. It was concluded that the extract possesses immune-bioactive. Administration of Nyctanthes arbor-tristis Linn leaves and  flowers chloroform extracts orally for 27 days caused a significant reduction in LPO,SGPT, SGOT, Alk phos, cholesterol and triglyceride level on extracts treated STZ diabetic rats, compared to diabetic control rats.

Materials and Methods

Alkaloid extraction

Procedure:

To isolate alkaloids from flowers, the dried and powered flower is extracted with pet ether     ( or hexane, colemans etc.) first. This removes fats, oils, terpenes, waxes etc. this extract is discarded.

The material is now subjected to an alcohol extraction, e.g. with ethanol. This extract is evaporated to leave the crude alkaloids mixture.

This extract is partitioned between an diluted aq. Tartaric acid solution and ethyl acetate or any other suitable organic solvent. Other acid like citric acid can be used, and other solvents may substitute here. The ethyl acetate layer contains neutral and weakly basic alkaloids. Evaporate the solvent to isolate them.

The aq. layer is neutralized with NH3 or Na2CO3 and again extracted with ethyl acetate. The organic layer now contains basic alkaloids, while the aq. Layer contains quaternary ammonium ions.

Many alkaloids can be isolated directly from the alcoholic extract by chromatographic methods. This is a separation which works well for tropane alkaloids (atropine, cocaine, scopolamine).

The alcohol extract is fractionated by column  chromatography on SiO2, solvent chloroform followed by chloroform with rising methanol content. This separates lipids and terpenes from the crude alkaloid fraction. The alkaloid fraction is again chromatographed (SiO2; CHCl3 : MeOH =10:1) to isolate the pure alkaloids.

Results:

Using Information

Leaves are antibilious, cholagogue, diaphoretic, diuretic and expectorant; used in bilious fever and rheumatism. Juice is a safe purgative for infants; given to children for the expulsion of round and thread-worms; with honey the juice is given in chronic fever. Decoction of the leaves is given for sciatica. Bark of the plant is expectorant; cures bronchitis. A decoction of bark, leaves, roots and flowers is given in excessive diuresis and in enlarged spleen. The flowers are stomachic, carminative and astringent to the bowels and tonic to the hairs; lessen inflammation. The seeds are useful in piles and skin diseases. Powdered seeds are used as an application for scurfy affections of the scalp. The Chakma take stem extract against dysentery.

During toxicological study of the leaf extract on laboratory animals, it was found that it significantly increased the weight of the heart and the kidney, and decreased the weight of the liver of male rats, but increased the weight of the liver of the female rats. It also increased ovarian weight. It slightly increased the total sum of the weight of all male sex organs. It caused a gross histological change of the kidneys, the liver, thymus, lymph nodes and lachrymal gland. It also caused some slight morphological abnormalities of the lungs.

Antibacterial activity:

Infectious diseases are world’s leading cause of premature death. Resistance to antimicrobial agents is emerging in a wide variety of pathogens and multiple drug resistance is becoming common in diverse organisms such as Staphylococcus aureus, Staphylococcus epidermis, Salmonella typhi

Methanolic extract of leaves and flowers of Nyctanthes arbor- tristis Linn exhibited significant antibacterial activity against those bacteria. The chloroform extract and the isolated compound (NCS-2) from its flowers were found to have larvicidal activity against common filarial vector, Culex quin que fasciatus.

Conclusion:

Researchers (Sandhar et al., 2011) revealed that the main active constituents in Nyctanthes arbor- tristis Linn parts are 6 hydroxyloganin (arbortristoside –A, B and C) along with other glucosides. The major uses have been found on extracts of leaves including bitter tonic, digestive, laxative, diuretic, diaphoretic to cure malarial fever, sciatica, anthelmintic and as antidote to reptile venom. This plant has also been found to be used traditionally. Powdered stem bark are used traditionally in rheumatic joint pain and as expectorant. The flowers as such are bitter, astringent, carminative, stomachic and used in some of the ophthalmic purposes. The juice of the flowers is used to prevent graying of hairs and baldness.

References:

Sandhar, H. K.,Kaur, M., Kumar, B. Prasher, S. An update on Nyctanthes arbor-tristis Linn. Internationale Pharmaceutica Sciencia.2011; 1: 77-86

Khatune N. A.,Islam M.E.,Rahaman M.A.A., Masaddik M.A., Haque M.E. In-vivo cytotoxic evaluaton of new benzofuran derivative isolated from Nyctanthes arbortristis L.on Ehrlich Ascite carcinoma cells (EAC) in mice. Journal of Medical Science. 2003:3: 169-173