When to perform resistance testing?

In most developing countries antiviral agents are scarcely available, if at all. Determining resistance in this context is, therefore, not very meaningful. Nevertheless, it is useful to be familiar with the underlying principles.

In the West resistance is determined:

(1) in a pregnant woman before initiating or after failure of therapy when a new treatment is being considered,

(2) upon initiating antiviral therapy in a child,

(3) after failure of therapy when a modification of the therapy is being considered. It should be considered

(4) for an as yet untreated patient before the start of therapy, especially if this is at the time of seroconversion. Viral mutants can in fact “disappear” after the acute phase (they are less easily detected) as they have a lower fitness, in the Darwinian sense of the word. If resistance is not determined in the acute phase of the disease, it is nevertheless advisable to store plasma so that such an investigation can be carried out later.

In (5) post-exposure prophylaxis an attempt should be made to get a sample from the index case.

In each case one should not wait until the results of the resistance determination are known before starting treatment.

However, when the results become available, treatment can be adjusted as necessary.

Techniques

Tests for resistance determination should be carried out with viral RNA derived from plasma, before therapy is stopped and before starting new therapy. It is only possible if there is a detectable viral load (preferably > 1000 RNA copies/ml plasma).

Genotype techniques

• Sequencing: amplification by RT-PCR and automatic sequencing. There is significant interlaboratory variability. In half of the laboratories it has been found that at least 25% of the virus population must consist of mutants before they can be detected.

• LIPA: Line Probe Assay for RT. Detects better some mixed virus populations but can miss certain mutations.

Phenotype techniques

• Virus culture: difficult and time consuming.

• Recombinant virus: the RT and Protease genes of the virus that is to be tested are integrated in a laboratory virus from which these genes have been deleted. This virus is then cultured. With this technique 25% of the virus population must also consist of mutants before any difference in sensitivity can be detected.

• Virtual phenotype: sequencing of the mutant virus and use of a software program to predict sensitivity/resistance.

Recommendations for the simultaneous administration of antiretroviral drugs with rifampin or rifabutin
Drug	With rifabutin	With rifampin	Comments
Saquinavir Hard gelatin capsules Soft gelatin capsules	Possible, if ritonavir is included in the regimen. Probable (dose: rifabutin 600 mg qd or 2-3 times/week) or 150 mg qd or 2-3 times/week.	Possible, if ritonavir is included in the regimen. Possible, if ritonavir is included in the regimen.	Pharmacokinetic data and clinical experience are limited. (dose: saquinavir 400 mg bid + ritonavir 400 mg bid) Pharmacokinetic data and clinical experience are limited. The co-administration of saquinavir + rifampin is not recommended without ritonavir.
Ritonavir	Probable (rifabutin: 150 mg 2-3 times/week)	Probable	Pharmacokinetic data and clinical experience on the co-administration of ritonavir + rifampin are limited.
Indinavir	Yes	No	Clinical data are limited but favourable. (dose indinavir: 800 mg tid or 1000 mg tid + rifabutin 150 mg qd or 300 mg 2-3 times per week).
Nelfinavir	Yes	No	Clinical data are limited but favourable. (dose nelfinavir: 750 mg tid or 1250 mg bid or 1000 mg tid + rifabutin 150 mg qd or 300 mg 2-3 times per week).
Amprenavir	Yes	No	No clinical experience, but should be possible with the normal dose of rifabutin.
Nevirapine	Yes	No	No published clinical experience. The combination would be possible with the normal dose of rifabutin.
Delavirdine	No	No
Efavirenz	Probable The combination is possible. (rifabutin: 450 mg or 600 mg qd, or 600 mg 2-3 times/week).	Probable. The combination is possible with the normal dose of rifampin + efavirenz 600 mg or 800 mg qd.

A final solution is expected from an AIDS vaccine. A number of theoretical approaches are being tested. On the one hand attempts are being made to find a vaccine that prevents infection itself, while on the other hand a vaccine that is administered to persons who are already infected and that influences the course of the infection in the good sense would be very useful. As AIDS vaccines are at present still in an experimental stage, prevention and control of the infection should be concentrated on three levels: sexual transmission, prevention of transmission via contaminated blood or needles, and mother-to-child transmission.

Information for the general population and for particular risk groups, counselling of seropositive persons, condom distribution (prostitutes), promotion of use and availability of condoms, counselling prostitutes, detection and treatment of other sexually transmitted diseases (STDs). In a number of countries it is the custom that after the death of the husband, the wife marries a brother or another member of the deceased’s family. This of course promotes transmission. In many countries the condom is still a very sensitive topic (taboo in public debates or even in private conversations within couples or family). One of the first actions to be taken in the campaign against AIDS in Thailand was the demystifying of the condom by all kinds of educational and promotional campaigns. In contrast to what is sometimes said, the promotion of condom use in tropical countries is not a hopeless task. It is being investigated whether a virocidal cream applied to the vagina offers some protection (so that women no longer have to depend on the good-will of their male partners). Circumcision of the man brings about a 2.5-fold decrease in the risk of HIV transmission from woman to man. It must be emphasised that circumcision does not give absolute protection.

Screening of blood for transfusion, limitation of blood transfusions and injections, strict sterilization of needles, syringes, etc. The intravenous drug abuse problem does not have the same proportions in Africa as it does in developed countries, though it is quite substantial in Southeast Asia. Medical personnel should avoid accidental contact with blood by wearing gloves, by not recapping needles, by putting needles immediately after use into containers with large openings, and by wearing protective clothing, masks, gloves and glasses when assisting childbirth. Hands should be washed with soap and water after contact with body fluids. Correct techniques should (of course!) be used in surgical operations. For example, during a surgical operation the handing over of a sharp instrument such as a scalpel should take place not from hand to hand, but the instrument should first be laid down, after which the next person picks it up. Making sterile needles and syringes available to drug users, as well as methadone projects, have proved useful.

Not all children born to seropositive mothers will be infected. Why some children are infected and others are not is a topic of intensive research. Seropositive women are advised to avoid conception. However, there are many women or married couples who do want a child to symbolically overcome death via their progeny. Breast-feeding should be discouraged if there is an alternative, though the latter is often not the case. Perinatal administration of Retrovir® definitely lowers the risk of transmission. In an American-European study in which Retrovir® was given IV to the mother throughout the third trimester of pregnancy and during childbirth and to the child for 4 weeks a decrease in transmission from 22% to 8% was observed. Shorter schemes with Retrovir® are also effective. Retrovir® appears to be tolerated very well by babies, with only minimal side-effects. In Third World countries Retrovir® will often be beyond the scope of many patients. Perinatal transmission can be reduced by 50% by a single administration of 200 mg nevirapine (Viramune®) to the mother during childbirth and by one dose (2 mg/kg) to the baby within the first 3 days of life. This should be a cheap strategy in Third World countries for reducing intra-partum transmission and transfer via the early maternal milk. Elective Caesarean section carries a significantly lower risk of transmission (about 10%). The advantage of a Caesarean section diminishes when the amniochorionic membranes are broken and/or the mother is already in labour. Combining prophylactic Retrovir® with elective Caesarean section can reduce the risk of vertical transmission to about 2%. The possible place of HAART in this setting is still not clear. Episiotomy, internal monitoring (in any case a luxury), forceps and vacuum extraction are best avoided in vaginal childbirth. After birth, the child should be gently aspirated, the eyes and nose flushed and given a quick bath to remove all blood. Optimal nutrition with enough vitamin A before giving birth is advised (the possibly inhibitory effect of certain retinoids on HIV is being investigated).

These viruses are filamentous in structure and are therefore known as filoviruses. Infections with these viruses have a very high case-fatality ratio. Fortunately, infections are not common, but if they occur, they tend to be dramatic.

Cholera is an acute infectious disease, characterised by profuse watery diarrhoea. It is caused by a Gram-negative bacterium: Vibrio cholerae O1 (the characters O1 indicate the serogroup). It is a very small, motile, curved bacterium (vibrio is the Greek word for comma). There is a single polar flagellum. Various subtypes exist, with classification according to biological and biochemical behaviour (biotypes) and serological characteristics (serotypes). Analysis of rRNA-genes (ribotyping), electrophoretic typing of multiple enzymes (zymovars) and bacteriophage typing are used in epidemiological research. Until 1992 it was thought that bacteria causing cholera must belong to V. cholerae, serogroup O1 and that they must be toxicogenic (must possess and express the genes for toxins). It was known that non-O1 Vibrio cholerae could sometimes cause mild gastro-enteritis or even septicaemia in immunodepression, but not cholera. In October 1992 in Madras (India), a mutated pathogenic bacterium (a new serogroup) was discovered. This new bacterium also causes cholera. The isolate was given the name Vibrio cholerae O139, nicknamed Bengali.

In August 2000 the whole genome (2 circular chromosomes) of V. cholerae O1, biotype El Tor was charted and published. Antibiotic resistance genes in V. cholerae are often positioned on plasmids and can be transmitted to vibrios from non-pathogenic intestinal flora. There are arguments to suggest that V. cholerae recently isolated from the intestine of a patient is much more infectious than bacteria which have been in the outside world for a long time.

Biotypes

There are 2 biotypes: classic Vibrio cholerae and V. cholerae biotype El Tor. Biotype El Tor agglutinates chicken erythrocytes and causes lysis of sheep erythrocytes, unlike the classic biotype. The name El Tor originates from the Egyptian/Saudi border town and quarantine camp El Tor in the Sinai dessert, where the bacterium was isolated for the first time in 1905 (during the 6^th pandemic) from an asymptomatic Hajj pilgrim from Mecca. The importance of this germ was long disputed (until 1961). At present El Tor has replaced the classic variant in most places, except in the Ganges and Brahmaputra delta. El Tor may also survive longer in the environment, is less dependent on transmission via water and produces more asymptomatic infections (symptomatic/asymptomatic infections = 1/90).

Serotypes

V. cholerae O1 may have 2 different antigens on its membrane. If only the first antigen is present, the bacterium is known as serotype Inaba. If only the second antigen is present, the bacterium is known as serotype Ogawa. If both antigens are present, the name Hikojima is given. Serotype shift seldom occurs (from Ogawa to Inaba and vice versa). The difference between these serotypes is only of importance for epidemiological studies. For example: in 1991 all cases of cholera in South America were caused by toxin-producing Vibrio cholerae, serogroup O1, biotype El Tor, serotype Inaba. The cholera epidemic in the Rwandan Hutu refugees in Zaire (July and August 1994) was caused by El Tor, serotype Ogawa. These bacteria were resistant to tetracyclines, cotrimoxazole, chloramphenicol and ampicillin.

Cholera has always been endemic in India and Bangladesh, in the huge delta formed by the confluence of the Ganges, Brahmaputra, Jamuna and Meghna rivers. Probably there was no cholera in Europe or America before the 19^th century. Between 1817 and 1923 there were various great pandemics, probably caused by the classic V. cholerae (there is no certainty as to the exact strain). The first pandemic which started in 1817 did not reach Western Europe. In 1829 the bacterium was introduced into the countries around the Persian Gulf via a British army unit stationed in India. From Iran the infection spread to Iraq, Syria, Georgia and Astrakhan (north of the Black Sea). It then travelled towards Odessa, Moscow, Vienna, Warsaw and Hamburg reaching England via the port of Sunderland. The first cases in London were seen in February 1832. The third pandemic merged with the second and was amplified by the miserable conditions during the Crimean war. The pathogen was discovered in 1884 by Robert Koch during the fifth pandemic (first work in 1883 in Alexandria, Egypt, confirmation followed by research in India in 1884, with isolation of the bacterium in culture). In fact the bacterium had already been described in 1849 by Pouchet and in 1854 by Filippo Pacini, an Italian physician. However, the latter’s work on this was not known outside Italy (he was known abroad due to his description of Pacini’s corpuscules, the pressure receptors in the skin). The germ theory and in particular the work of Koch were attacked by Pettenkorfer and his student Emmerich, who each drank a vial filled with bacteria as proof against the role of V. cholerae. Amazingly, Pettenkorfer did not then get cholera, but Emmerich suffered severe diarrhoea for 48 hours. When each pandemic began and ended is rather unclear. There was cholera in Belgium in 1832, 1848, 1854, 1859, 1866 and 1892. In 1866, 1 Belgian in 100 died of cholera.

Cholera pandemics since 1817
Number	Years	Origin	Pathogen
1	1817-1823	India	?
2	1829-1851	India	?
3	1852-1859	India	?
4	1863-1879	India	?
5	1881-1896	India	V. cholerae O1, classic
6	1899 -1923	India	V. cholerae O1, classic
7	1961 to present	Sulawesi	V. cholerae O1, El Tor
8	1992 to present	Madras, India	V. cholerae O139

After the sixth pandemic there was a strange silence for about 40 years, for which no good explanation exists. The seventh pandemic was caused by El Tor. It started in 1961 in Celebes (Sulawesi), Indonesia, reached India in 1964 and Africa in 1970. In 2 years the infection passed through 29 African countries. In 1973 it arrived in the Gulf of Mexico. Early in 1991 the infection spread rapidly in Peru. In 3 weeks there were 30,000 cases. The bacterium then spread further into South America, causing 360,000 cases within the year. In the summer of 1992 a second, less severe outbreak occurred. Nevertheless by August 1992 "only" 5,000 deaths had been reported (from an estimated total of 600,000 cases), thanks to the wide-spread use of rehydration therapies. The case-fatality ratio varied depending on the region. After 1993 the disease assumed an endemic character in several countries, sometimes with local outbreaks. At the end of 1993 the cumulative total amounted to 900,000 cases in three years (1991-1993), with a cumulative mortality of 8,000. According to one hypothesis cholera bacteria infected the marine plankton off the Peruvian coast via the ballast water from a Chinese freighter. The possible role of changes in the nutrient-rich von Humboldt current is still unclear.

About 80% of the cholera in 1997 occurred in Africa, chiefly in the horn of Africa (118,000 cases were reported officially). The increase in cholera in this region followed heavy rains and flooding (possibly associated with the El Niño weather phenomenon).

Since 1992 V. cholerae O139 is recognised as a cause of a disease which is clinically identical to classic cholera, but which also occurs frequently in adults. Classic cholera in India, on the other hand, is common in children. There is no cross immunity with V. cholerae O1. Bacteria of the 0139 serogroup have a polysaccharide capsule (unlike V. cholerae O1), which may explain the increased risk of septicaemia. In the following years this new serogroup spread across Bangladesh, India, Pakistan and Southeast Asia. By the end of March 1993 more than 100,000 cases had been reported in Bangladesh. If further spread continues, as with the earlier epidemics, it will be possible to refer to this as the beginning of the eighth pandemic. It was observed in India that, after the first spread of V. cholerae O139, new variants (clones) of V. cholerae O1 El Tor once more gained the upper hand. Cholera also surfaces regularly in Madagascar. From the beginning of December 1999 until the end of February 2000 more than 12,400 cases were reported. The disease can thus certainly not be regarded as an entity which only existed “in the past”.

In the first half of the nineteenth century a cholera epidemic occurred in London. The physician John Snow, already well-known in 1853 as anaesthetist to Queen Victoria (chloroform during childbirth), had a special interest in cholera. In 1854 he examined the various families presenting cases and calculated that the mortality in the houses which were supplied with water by the Southwark and Vauxhall Water Company was 31/1000 houses. This was 8.5 times higher than in houses supplied by the rival Lambeth Company. Although neither of the two companies offered purified water, the first company took its water from the Thames near London Bridge, downstream from the city sewage outlets while the second company pumped its water upstream from the city at Thames Ditton. In 1849 there had been no difference in mortality between the families which received water from Lambeth or Southwark. Before 1851 the Lambeth Company drew its water from a highly contaminated stretch of the Thames near Hungerford Market. It was this spectacular change (1849 compared to 1854) which made Snow conclude that contaminated water had a causal connection with cholera. Although both companies delivered water in the same streets, the water used in any particular house could be identified by its salt content (London Bridge is closer to the sea and its water is more salty than that at Thames Ditton). Adding silver nitrate leads to precipitation of silver chloride, proportionate to the amount of salt in the water. This was the basis of a simple test which could be carried out in every house.

During a particular outbreak he mapped the houses where cholera occurred. On the map spatial clustering around the Broad Street pump could be seen. The houses where cases occurred were closer to the Broad Street pump than to other water pumps. People usually took their water from the nearest pump. There was no similar clustering in front of the sewage outlets, which argued against the miasma theory which was popular at that time (fumes from the sewers). He noticed that families where cholera occurred, used water from a particular public water pump in Broad Street, St James’s parish, Soho. Families which were spared, obtained their water elsewhere (there were five water pumps in the area). After the pump had been turned off (the handle was removed), the epidemic stopped (although the epidemic was already diminishing before the pump was made unusable). Careful inspection showed broken masonry in the well. A few metres further up was a sewer from which water was overflowing into the well. It was quite a new concept to use maps for epidemiological purposes.

Similar findings were made in Hamburg in 1890. The incidence of cholera was 34/1000 in Hamburg, where the drinking water was drawn from the river Elbe, and 3.9/1000 for the surrounding areas where other sources were used. In Altona, downstream from Hamburg, contaminated water was also taken from the Elbe, but there was less cholera. How could this be explained? If anything, more cholera would be expected in Altona. The difference was that in Altona the water was first filtered slowly through sand before being supplied for consumption. These observations led to attempts to provide cities with clean drinking water and to construct adequate sewers. Cholera had an enormous effect on the development of the principles of public health and was the basis for the so-called “sanitary revolution” at the end of the 19^th century. Cholera was the first disease for which surveillance was set up and because of this the disease has code number 001 in the international classification list of diseases.

Cholera is spread by the faeco-oral route, via contaminated water and food. Asymptomatic infections are common, especially in case of El Tor. People excrete bacteria for about 10 days. This is sufficient time to ensure continued contamination of the environment. Chronic carriers are very rare. In third world countries many people have no chlorinated, filtered, treated, pure drinking water. The lack of good toilets and sewers leads to contamination of the surface or ground water. Too often untreated sewage water is still poured into surface water. Sometimes sewage pipes and drinking water pipes are laid in the same trench, which may result in contamination if there are leaks or greatly varying water pressures in the pipes. If drinking water is infected in this way, bacteriological checks of the drinking water when it leaves the pumping station will not show anything amiss. In houses, drinking water containers with a wide opening often become infected, because people are inclined to scoop up water in their (dirty) hands. Containers with a small spout, from which water must be poured, are much safer.

There is also direct transmission from person to person, but it is rare. The number of bacteria on dirty hands is usually lower than the minimum infectious dose necessary for direct transmission (>10⁵ bacteria). A large inoculum is necessary to cause infection. Health workers who respect basic hygiene are at extremely low risk. Filter feeders such as mussels or oysters concentrate the bacteria in their bodies. These molluscs were the source of a recent outbreak in Hong Kong. When the organisms adhere to food particles (e.g. the chitin of crustaceans) and in the case of hypochlorhydria, lack of gastric acid due to gastric surgery, antacids, anti-ulcer drugs or atrophic gastritis, the number of organisms needed to trigger infection is much smaller. Food may be infected by dirty hands during or after preparation. The bacteria can survive and reproduce in food such as cereals, rice or lentils and crustaceans. This intermediate replication step is very important. If someone dies of cholera and a meal is made for the mourners by the persons who have washed the corpse, the risk of further transmission is very real. The bacteria are very sensitive to drying out, sunlight and acid. Meals which contain acid, e.g. tomatoes and/or lemon, are much less dangerous than neutral or alkaline meals. Vegetables and fruit on the market are often sprayed with water to make them appear fresher and more attractive. If this is done with infected water, transmission may occur.

Humans are the only vertebrate hosts. Vibrio cholerae can survive long-term and probably permanently in brackish water, especially if there is a a neutral or slightly alkaline pH and the water contains some minerals and organic material. The bacteria are concentrated in phytoplankton (certain algae) and zooplankton which live in this water. Among the latter, copepods, a group of crustaceans, are important. V. cholerae O1 produces chitinase, an enzyme which can break down chitin. This is a structural protein of the exoskeletons of invertebrates. The full importance of this chitinase has still to become clear.

Vibrio cholerae is also found in association with a cyanobacterium (Anabaena variabilis). Vibrio cholerae has similar interactions with marine diatoms (Skeletonema costatum), phyaeophytes (Ascophyllum nodosum) and copepods. The bacteria also flourish in fresh water where plants such as water hyacinth are growing. The impact of specific ecological circumstances which might lead to a sudden increase in plankton (algal bloom) needs to be studied further. Cholera is clearly seasonal. A chronic aquatic reservoir is likely and this might be independent of continuous human faecal pollution. V. cholerae excreted by humans can be cultured in the laboratory. These bacteria may assume a living form which cannot be cultured in vitro and which multiply in the environment. However, those bacteria are not dead since they multiply when instilled in a rabbit's ileum (rabbit ileal loop model). It may revert to a replicating form in its natural environment when there are favourable environmental factors and this has important epidemiological implications. The living, but non-reproducing form of V. cholerae can probably cause disease. Traditional culture methods for tracing V. cholerae in water miss these “dormant” bacteria. Tests based on fluorescent antibodies may offer a practical solution.

Half empty cargo ships take water in their ballast tanks in order to lie deeper and with more stability in sea water. This ballast water of course contains micro-organisms. In the past potentially infected ballast water was pumped out again when the ship came into harbour at its destination. In view of the importance of an aquatic cholera reservoir, freight ships have to empty and refill their ballast water tanks while in open sea, and not when they come into harbour.

The bacterium multiplies in the small intestine, where it adheres to the mucosal brush border. The bacterium is not invasive, in other words it does not penetrate the intestinal wall or pass into the blood. It excretes a very powerful toxin, which inhibits the absorption of fluid from the intestinal lumen and causes active fluid secretion towards the lumen. This fluid is isotonic, ion-rich and protein free. There are no intestinal ulcerations and the faeces do not contain blood. There is little if any fever. There is no tenesmus. The faeces contain significant amounts of sodium, potassium and bicarbonate. Because of this the intestinal content is slightly alkaline (V. cholerae thrives best in a slightly alkaline environment and is therefore producing the conditions which are optimal for its own survival). The loss of large amounts of alkaline faeces results in metabolic acidosis. People with blood group O have an equal risk of infection but are at a significantly higher risk of clinically severe cholera if they become infected. The reason is unknown.

Vibrio cholerae produces several toxins: an enterotoxin, the zona occludens toxin (zot) and the accessory cholera enterotoxin (ace). The role of the two latter toxins is unclear. The enterotoxin of V. cholerae consists of 2 parts: A (active) and B (binding). The polypeptides of part B (a pentamer of five identical subunits) bind to a receptor (G_m1 ganglioside, a glycolipid ) on the epithelium of the small intestine, after which part A (a monomer) can penetrate the cell. The latter protein binds covalently to an intracellular protein (G_s-protein; s for stimulatory) which irreversibly activates it, leading to the persistent stimulation of another intracellular enzyme, adenylate cyclase. This latter enzyme promotes an increase in the intracellular cyclic-AMP, which inhibits salt absorption and promotes active chloride excretion. Water and potassium bicarbonate passively follow the chloride. In the end there is an overall water loss to the intestinal lumen. The colon cannot absorb the large amount of fluid quickly enough and diarrhoea follows. The toxic A-subunit also has other effects such as disturbing the expression of some genes, increasing inflammatory cytokines and inhibiting antigen presentation by macrophages. On the other hand, the B-subunits of cholera toxin have anti-inflammatory properties. These are under intense study at present for possible therapeutic use in immune abnormalities.

The in-vivo mechanism is probably more complicated. Cholera toxin also stimulates the nervous system in the intestinal wall (myenteric plexus), resulting in the release of 5-hydroxytryptamine (serotonin) from the enterochromaffin cells. This results in additional fluid loss to the lumen. Granisetron, a 5-HT3 receptor blocker, partially reverses this effect. More research is needed to determine the role of this mechanism in the physiopathology.

Enterotoxin  stimulation of G-proteins  stimulation of adenylate cyclase  increase in c-AMP  chlorine excretion to the intestinal lumen

The incubation period is brief: 1 to 5 days (usually 2 days). Asymptomatic infections are common, but chronic carriers are very rare. Sometimes there is an initial transient fever. Massive watery diarrhoea starts suddenly. The faeces very rapidly look like water in which rice has been boiled: watery with flakes of mucus. The faeces have a fish-like smell. The volume of faeces may rise to 500 ml per hour. Vomiting is common, but abdominal cramps are unusual. The onset of thirst, oliguria or anuria and weakness is rapid. In a short time the patient develops severe dehydration and can die within 24 hours. In other cases the diarrhoea is less severe, especially with infections with El Tor. As the patient’s condition deteriorates, hoarseness of the voice and temporary deafness are often observed.

The signs of dehydration are thirst, dry mouth and lips, hollow eyes and sunken fontanel in children. The skin turgor diminishes. The skin becomes wrinkled (washerwoman’s hands). The voice becomes weak and hoarse, the pulse quickens and is difficult to feel. Blood pressure falls. There is little or no urine production. Respiration becomes faster due to metabolic acidosis secondary to loss of bicarbonate in the faeces (bicarbonate is alkaline). This acidosis causes vomiting and muscle cramps. There is also significant potassium loss in the faeces. If rehydration is carried out using fluid without potassium, severe hypokalaemia may result. Nevertheless, quite often normokalaemia is found, together with an increased anion gap. The increase in the anions (= negative ions) is multifactorial due to the hyperproteinaemia (haemoconcentration), hyperphosphataemia (internal shifts and renal failure) and lactate acidosis (shock). Ketones play little if any role. The mortality from classic cholera may reach 50 %, but can be brought down to <>

Cholera should be suspected in acute massive rice-water diarrhoea, certainly if there have been several cases in a short time (epidemic). The clinical picture of severe cholera is so spectacular that differential diagnosis does not present many difficulties. Milder cholera may be similar to other forms of gastro-enteritis (but not to dysentery). A child above the age of five years who develops acute dehydration, or dies as the result of acute diarrhoea, is always suggestive for cholera.

The vibrios are very small and can best be seen in a fresh faecal specimen with the help of dark field microscopy. There is characteristic motility ("star shooting") which stops immediately after adding anti-O1 antiserum. This does not give any information on possible toxin production. Confirmation is best made via a bacteriological culture. Culturing should preferably be on a special medium in a bacteriology lab, e.g. TCBS-agar [= Thiosulphate-Citrate-Bile salts-Sucrose], polymyxin mannose tellurite agar (PMT) or an other selective medium. In order to identify the serogroup and the serotype one subsequently finds out to which antibodies (antiserum) the colonies obtained exhibit an agglutination reaction. It is also possible to find out whether the vibrios are toxicogenic (produce toxin). Definitive identification is made in a reference laboratory.

Specimens may be transported in a transport medium, e.g. Cary-Blair. This is a kind of mild alkaline gelatine in seawater in which the bacteria will survive for 4 weeks. If it is not available, a filter paper can be soaked with faeces and transported in an airtight bag to a well-equipped laboratory. A sample treated in this way remains usable for 1 week, but the recommendation is "the faster the analysis, the more reliable". Blotting paper, soaked with liquid faeces and if possible placed in a 1% saline solution, can be kept for several weeks at 37 (not in the freezer). This is useful if there are initial transport problems. Nevertheless it is better to have a fresh faecal specimen. For specimens from the environment or from food, in which the number of bacteria is much lower than in faeces, enrichment is necessary. The specimen can be incubated for 8 hours in alkaline peptone water, after which a TCBS agar is used.

About 10 days after infection with V. cholerae O1 the patient produces vibrocidal antibodies. They start diminishing after only one month and disappear within the year. Antibodies against cholera toxin are produced more slowly and remain for years. However, these cross-react with enterotoxin produced by ETEC bacteria [enterotoxic Escherichia coli]. The immune response to V. cholerae O139 is not well understood. The detection of antibodies is not important for the urgent care of the individual patient, but does permit retrospective diagnosis.

Rehydration is essential and must be instituted as soon as possible. Two phases are distinguished. First it is important to replenish what has been lost in the previous hours or days. Then one must compensate the persistent fluid loss (e.g. the amount of fluid that is lost every hour). In mild cholera without vomiting oral rehydration may suffice. In severe forms IV fluids should be administered.

There are several possible compositions of rehydration fluids. Solutions containing salt, sugar, potassium and bicarbonate are recommended. Acetate is also used. Lactate is also good because it is converted in the body to bicarbonate. In cholera it is preferable to use Ringer’s lactate ( = Hartmann’s solution). Normal physiological saline is second choice because it does not correct the acidosis nor does it contain potassium. Severe hypokalaemia may occur, with cardiac arrhythmias, kidney damage, paralytic ileus and significant muscle weakness with reduced or absent tendon reflexes. Dextrose (= glucose) 5 % without electrolytes is not advised as a rehydration fluid. A reminder: 1 gr KCl = 13 mEq KCl.

So: Ringer’s lactate >> physiological saline; not glucose infusion if there is an alternative

In severe cholera (fluid loss > 10 % of weight) the missing fluids should be administered quickly, e.g. 6 litres over 4 hours for a patient weighing 60 kg. The first 3 litres may each be administered in 10 minutes (total therefore 30 minutes). After administration of the lost volume, the losses are compensated for further IV and/or PO fluids (faeces volume + urine volume + 500 ml). Vomiting may make oral administration of fluids difficult. Generally a total of 6 to 10 litres per patient is necessary. When patients start to drink and do not vomit anymore, it is best to leave their IV lines in place for a while until one is sure that rehydration will not pose any more problems.

Special cholera beds are useful: they have a central opening to allow the liquid faeces to pass through, and they can be collected in a bucket. This makes it possible to quickly determine the amount of fluid loss. During an epidemic people who can still hold themselves upright can simply sit on a bucket and try to drink as much ORS [oral rehydration solution] as possible. Children quickly develop convulsions and coma. It is important that hypoglycaemia should be considered. For an adult 50 ml of a 50% glucose solution is given IV, for a child 2-4 ml/kg 25% glucose or 10 ml/kg of a 10% glucose solution.

Antibiotics are useful because they reduce the duration and thus the total volume of the diarrhoea. They are not essential, however, and resistance often occurs. Classic V. cholerae is usually sensitive to tetracyclines. The dose of tetracycline is 500 mg x 4 daily PO for 2 to 3 days. Parenteral antibiotics are of no value (the bacteria are not invasive). Alternatives are doxycycline 300 mg once or erythromycin, cotrimoxazole and chloramphenicol. Single-dose ciprofloxacin is also effective. V. cholerae O139 is often resistant to cotrimoxazole (sulphamethoxazole-trimethoprim) but sensitive to tetracyclines. This resistance to cotrimoxazole is determined by a certain genetic element (the SXT [sulphamethoxazole] element). Antiperistaltic drugs such as loperamide may cause accumulation of fluid in the intestinal lumen with unfavourable consequences.

In the West nowadays a patient with cholera will remain a sporadic case. In developing countries one case can lead to several secondary cases. It is not necessary to wear special masks, aprons or gloves, but washing hands (hospital staff, family, visitors) should be obligatory. The contamination of clothing and bedding is unavoidable. Boiling in water for five minutes is sufficient for disinfection. Mattresses and blankets can be dried in the sun. It is better to do this before washing them, to prevent infection of the washing area.

After surviving cholera a patient is probably immune for more than 3 years. No cross-immunity between V. cholerae O1 and V. cholerae O139 is seen, although they produce the same toxin. Immunity relies on antibodies in the intestinal lumen (the bacteria are not invasive). Babies which are being breast-fed receive protective antibodies in their mother’s milk. Vaccination with dead V. cholerae bacteria (IM administration) does not lead to the formation of protective antibodies in the intestinal lumen. Advice to vaccinate was discontinued in 1972 by the WHO [World Health Organisation]. New oral vaccines are being tested. Vaccination, mass chemoprophylaxis and cordon sanitaire (= restrictions on travel and trade) are not effective in preventing or limiting outbreaks.

Mass chemoprophylaxis is not effective because (1) the infection spreads faster than the organisation of drug distribution, (2) the effect of a drug only lasts 2 days, after which re-infection may occur, (3) the whole population needs to be treated simultaneously and people should then be isolated and (4) it is difficult to convince asymptomatic people to take a drug. Selective chemoprophylaxis of nearby contact persons can be given, certainly if there appear to be multiple secondary cases (the role of monitoring is important here).

Correct eating and drinking habits, safe stool habits and personal hygiene are the most effective means for individuals to limit their risk of cholera. Improved sanitation is the pre-eminent method of eliminating cholera and many other faeco-orally transmitted infections. This is directly linked, however, to the degree of poverty in a region. Boiling drinking water is often difficult since fuel may be scarce and expensive. Since a significant proportion of Vibrio cholerae can adhere to plankton, the drinking water can be filtered through a fine cloth, which removes both plankton and a lot of bacteria in a single operation. This is of course less effective than obtaining water from a clean pipe or pump, but it is cheaper. Chlorination of drinking water may be important (piped water or via water trucks). This is difficult to accomplish in rural areas. Chlorination is much less effective if the water is turbid due to organic debris.

Eating raw fish, shellfish (e.g. oysters, mussels) and crustaceans (such as crabs, shrimps) should be avoided. Washing hands is important for transmission control within a household. Infected faeces should not be disposed of in a poorly functioning drain (hospital: e.g. in pit with unslaked lime). When large groups of people come together (funerals, festivals, etc.) there should be latrines with facilities for washing hands.

An attempt must be made to trace the source of small, local outbreaks (see John Snow). Infected water is the chief suspect in a sudden, local epidemic, while in isolated cases the cause should be sought in infected food. This is of course not an absolute rule. Food cooked by street vendors and in restaurants poses specific problems. Flies probably play an underestimated part in transmission, but their numbers also reflect the sanitary conditions in a region.

The following points should be emphasised during information campaigns:

• Drink only clean water (boiled or chlorinated)

• Cook food completely and eat it while it is hot

• Avoid uncooked food, unless it can be peeled

• Wash hands after a bowel movement

• Wash hands before preparing food

• Wash hands before eating

• Correct use of a good latrine (also for children)

• With correct treatment cholera is rarely fatal

• If cholera is suspected medical help should be sought immediately

• In diarrhoea, give plenty of fluids (e.g. ORS)

• No cholera-vaccination (unless better vaccines are developed in the future)

In case of an epidemic, it is important to have a large stock of IV rehydration fluid available as well as the means of preparing large amounts of oral rehydration fluid. Normally such buffer stocks should be stored at various strategic points. The stocks for cholera treatment should not be segregated in storage, but should be rotated during normal use to avoid allowing large amounts to run out of date. As soon as an epidemic is suspected, use as much oral rehydration as possible so that stocks of the IV solutions last as long as possible. Cholera beds should be made ready. In a normal epidemic an attack rate of 0.2% can be taken as a rule of thumb (i.e. 200 cases can be expected in a population of 100,000). This is useful for estimating the size of stocks that will be needed. Sometimes the attack ratios are higher (e.g. the Rwanda-Zaire border in 1994).

During an epidemic one should consider

1. clean drinking water in sufficient amounts,

2. sanitary provisions (latrines + hand washing with soap ± hand desinfection with hypochlorite solution),

3. treatment of the patients

4. direct contact persons of the patient (e.g. mother of a child) may take chemoprophylaxis, but mass prophylaxis is not advisable.

5. The risk for a traveller who observes elementary hygiene is very small (1/500,000).

• Capsid: Composed of numerous repeating subunits. Viral genome is thus conserved in that a single protein can be transcribed many times to make the capsid.

• Capsomere: Clusters of polypeptides which, when completely assembled, form the capsid.

• Nucleocapsid: Genome together with capsid.

• Virion: Free viral particle, consisting of nucleocapsid, or nucleocapsid plus envelope.

• Envelope: Lipid bilayer, usually obtained from host cell.

• Symmetry: Three types

  • Cubic Symmetry: Icosahedral symmetry. 20 equilateral triangles.

  • Helical symmetry

  • Complex: No regular symmetry, as in poxvirus.

• Viral Conditions:

  • Alkaline kills all viruses.

  • Ether: Enveloped viruses are susceptible to ether. Poxviruses vary in susceptibility.

  • Enveloped viruses are generally less stabile than naked viruses.

• Host-cell Virus Interactions:

  • Productive Infection: Virus successfully completes replication cycle and produces progeny virions.

  • Abortive Infection: Virus enters the host-cell but cannot successfully complete replication.

    • Can result from a non-permissive host-cell, or because the virus is defective.

    • Abortive infections can become persistent infections.

  • Restrictive Infection: Cells are only transiently permissive, after which they become non-permissive. This results in transformation of the cell.

  • Latent Infection: Virus remains in a cell and does not replicate, as in HSV, VZV, CMV, EBV, and HIV.

• Replication Cycle:

  • Divided according to presence of infectious virions:

    • Eclipse Phase: Virus has entered cell, but no progeny virions are created yet.

    • Maturation Phase: Viral progeny are produced and infectious virions are again present.

  • According to biosynthesis sequence

    • Early Phase: Biosynthesis of prerequisite proteins, required before replication can take place, such as replicase enzymes or enzymes that inhibit host-cell synthesis.

    • Late Phase: Synthesis of structural capsid proteins, and actual replication of the genome.

• (+)-STRAND RNA: The RNA genome can directly serve as an mRNA, because it contains its own 5' cap and 3' Poly-A tail.

  • Isolated mRNA strands are infectious.

• (-)-STRAND RNA: They contain no cap and tail.

  • Isolated mRNA strands are not infectious.

  • They utilize a viral RNA-Dependent RNA-Polymerase to make (1) Subgenomic mRNA's (non-structural and structural proteins), and (2) a complementary (+) RNA genome.

  • The positive-strand RNA genome then serves as a template to make more minus-strand RNA's, to be packaged with the structural proteins.

• Polyproteins are often made by viruses, which are then cleaved, as eukaryotic machinery can't recognize a polycistronic mRNA.

• Double-Stranded DNA: Herpes, Papova, Adeno

  • Early Genes are transcribed by host cell RNA-Polymerase, to form early proteins.

  • These early proteins include a viral DNA-Dependent DNA Polymerase, which then forms progeny DNA.

  • After replication, late genes are transcribed and translated, forming structural proteins.