Sanjiv Rughooputh

Researcher

Molecular and Microbial Glycobiology Research Group

Graduate Centre in Biosciences

London W1 M8 JS.

Email: S.Rughooputh@wmin.ac.uk

Diphtheria has for centuries caused devastating epidemics. With the advent of mass immunisation in the developing world in the 1950’s the disease was regressing and was in the process of becoming extinct. In the early 1980’s the lowest level of cases was documented.

However, with the collapse of the Soviet Union in the late 1980’s, the disease peaked with more than 50,000 cases in Europe alone. About 3000 people were diagnosed with diphtheria in the Ukraine in 1993 as compared to just 1500 in 1992. Diphtheria is now documented in all regions of the world including Africa, South East Asia and South America. Diphtheria still remains problematic in 5 countries of the Former USSR: Tajikistan, Kyrystan, Georgia, Latvia and Ukraine

With the increase in international travel, migration of population to and from epidemic areas, especially with many Mauritians studying in Ukraine and also with the decline in immunity levels amongst populations highlight the need for better microbiological surveillance and greater clinical and laboratory awareness.

Diphtheria is an acute infectious disease caused by a bacterium called Corynebacterium diphtheriae ( C diphtheria). This disease affects the upper respiratory tract and very occasionally the skin. Virulent strains of C. diphtheriae produce a toxin that can damage heart and nervous tissues, although non-toxigenic strains can cause a localised infection. A diphtheria-like illness is sometimes caused by another toxin-producing bacterium Corynebacterium ulcerans.

Humans are the only known source of C. diphtheriae and the disease is spread by infected droplets or secretions from the nose and throat of a recent case or someone who, without any symptoms, is carrying the bacterium in their nose or throat. After an incubation period of usually between two and five days a new case may become generally unwell with a mild fever, headache, rapid pulse and sore throat. Swelling of the throat tissues may occur leading to breathing difficulties. The swelling is due to inflammation of the lining of the throat resulting in an exudate or 'membrane' which is typically pale Grey or white in colour and firmly stuck to the throat. Sometimes the infection is confined to the nose causing a foul smelling, bloodstained discharge. Progression of the illness then depends on whether or not the infecting strain of C. diphtheriae produces toxin and the amount of immunity already possessed by the patient from previous diphtheria immunisation. Milder cases will usually have an uneventful recovery once the throat membrane disappears after about a week. More serious infections are characterised by increasingly severe heart and nervous system complications, which develop after two to six weeks and can lead to collapse, paralysis, coma and death. About 5 to 10% of diphtheria cases die.

Chronic skin lesions due to diphtheria are unusual and occur particularly in the tropics. The lesions may be due to either toxigenic or non-toxigenic strains of C. diphtheriae. Spread of this infection is by direct contact with the infected lesions. The diagnosis and treatment of cases and the management of contacts is similar to diphtheria of the respiratory tract.

Most diphtheriae are usually isolated from throat swabs. However, isolation has also been documented in nose, skin or wound swabs. Generally, swabs are taken from the nose and throat of the patient suspected of having diphtheria, ideally before antibiotic treatment is started. Any wounds or skin lesions are also swabbed.

These swabs are sent to the Microbiology laboratory as soon as possible. For best results, the swabs are cultured on Blood agar and a selective medium like Hoyles Tellurite medium. Any Gram positive isolated from the selective media should be tested for pyrazinamidase activity and cystinase production to differentiate between the potentially toxigenic corynebacteria and other coryneforms. Full identification can also be done biochemically or by commercial kits like API Coryne, or Crystals from Becton –Dickinson. If C. diphtheriae is isolated then the bacterium must be tested to see whether or not it produces toxin as this will determine how the patient's illness is likely to progress and how much at risk are the patient's close contacts. In addition to swabs, a blood sample should also be collected from the patient before any antitoxin treatment is given, to measure their antibodies to diphtheria toxin; a second 'convalescent' blood sample should be collected about two weeks later to re-measure these antibodies.

Patients with diphtheria require urgent admission to hospital where they must be nursed in isolation. If diphtheria is confirmed then they must remain isolated until their treatment has been completed and negative cultures have been obtained from repeat nose and throat swabs. Treatment consists of an injection of antitoxin to counteract the effects of the toxin produced by C. diphtheriae and antibiotics, such as penicillin or erythromycin by injection and then by mouth for a total of seven to fourteen days, to kill the bacteria. Antitoxin and antibiotics are usually commenced before there is bacteriological confirmation of the infection. If necessary, action must be taken to assist the patient's breathing.

It is also recommended that people who have been in close contact with a case of diphtheria, especially a toxigenic strain in the previous 7 days must be screened and given prophylactic antibiotics. However, contacts in either a school classroom, workplace or hospital are at much lower risk.

All close contacts, whether or not they have received diphtheria immunisation in the past and irrespective of their nose and throat culture results, should be given a suitable antibiotic either as a single dose by injection or as a seven-to-ten day course by mouth. Those close contacts found to be carriers require a second throat swab after completing their antibiotics, to ensure they are now clear of carriage. A further ten-day course of antibiotics may be needed if carriage has not been completely eradicated. In addition, all contacts who have previously received diphtheria immunisation should be given a booster dose of vaccine unless their last dose of vaccine was within the previous 12 months. All unimmunised contacts should be given a full course of three injections of diphtheria vaccine. Contacts of cases due to non-toxigenic strains are not at risk and no further action is necessary.

( This article is only a general guideline, further information on patient management and treatment should be obtained from the local Clinical Microbiologist based at the Central Health Laboratory, Candos )

A throat swab was taken and blood was taken for Haematology and Biochemistry profiles.

The Haematology profile was normal with Haemoglobin of 13.6g/L, WBC count 6.1 X 10^9/L with a differential count of 57% Neutrophils, 32% Lymphocytes, 8% Monocytes, 2% Eosinophils and 1% Basophils. The platelets count was 291X10^9 and RBC count of 4.77X10^12/L. ESR was 5 mm/hr.

The Biochemistry profile was normal except for serum Chloride which was slightly lowered :

Sodium 137 mmol/L, Potassium 4.3 mmol/L chlodide 96 mmol/L( 98-111) Urea 4.5 mmol/L and Glucose 6.2 mmol/L.

The throat swab was cultured on 2 blood agar and 1 Hoyles plate. One of blood plate was incubated anerobically for 24 hrs (overnight) at 37*C, while the Hoyles plate was incubated at 37*C for 48hrs in air. The second blood plate was also incubated in air at 37C, but for only 24hrs. A Gram’s Smear was also done from the swab.

The Gram smear showed the presence of a few WBCs and Vincents organisms were absent.

The Blood plates read at 24hrs did not show any evidence of Beta Haemolytic Streptococcus.

After 48hrs incubation the Hoyles plate showed the presence of black colonies, which on Gram film were Gram Positive Rods. Further work was done in a class 1 cabinet to prevent aerosol being generated

The organism was further characterised biochemically and identified as Corynebacterium diphtheriae var gravis.

The isolate was sent for toxigenic testing and was fortunately non toxigenic.