WHO/CDR/95.4 Guidelines for the control of epidemics due to Shigella dysenteriae type 1
World Health OrganizationEmerging and other Communicable Diseases,Surveillance and Control
This document has been downloaded from the WHO/EMC Web site. Theoriginal cover pages and lists of participants are not included. Seehttp://www.who.int/emc for more information. World Health Organization This document is not a formal publication of the World Health Organization (WHO), and all rights are reserved by the Organization. The document may, however, be freely reviewed, abstracted, reproduced and translated, in part or in whole, but not for sale nor for use in conjunction with commercial purposes.
The views expressed in documents by named authors are solely theresponsibility of those authors. The mention of specific companies or specificmanufacturers' products does no imply that they are endorsed orrecommended by the World Health Organization in preference to others of asimilar nature that are not mentioned. TABLE OF CONTENTS Introduction.1 About Shigella dysenteriae type 1 (Sd1) .1 Other causes of dysentery.2 Prevention of infection with Shigella dysenteriae type 1 .2
Preventing spread of Shigella dysenteriae type 1 in health facilities .5
Disinfecting clothing and disposing of bodies .5
Preparation for Shigella dysenteriae type 1 epidemics .5
Use of antimicrobials when the supply is limited .7
Principal steps in the management of patients with dysentery caused by Shigella dysenteriae type 1 .10 Details of management of patients with dysentery caused by Shigella dysenteriae type 1 .12
When an effective oral antimicrobial is available. 13
When an effective antimicrobial is in limited supply .13
Role of the laboratory.15
Determine antimicrobial susceptibility of Shigella dysenteriae type 1 .15
After an outbreak .16 Acknowledgements.16
1. Sample public health messages for prevention of Shigella dysenteriae infection .17 2.
Rules for preparing food safely to prevent dysentery . 19
Building a ventilated improved pit latrine . 22
Collection and transport of stool specimens for of Shigella . 23
Supplies needed for laboratory identification of Shigella dysenteriae . 26
Laboratory identification of Shigella . 27
Antimicrobial susceptibility testing of Shigella . 33
Isolation and identification of Escherichia coli O157. 38
Treatment supplies for 100 persons in dysentery epidemics. 39
WHO Collaborating Centres for Shigella . 41
INTRODUCTION Shigella dysenteriae type 1 (Sd1) is an unusually virulent enteric pathogen that causes endemic orepidemic dysentery with high death rates. It is the only cause of large-scale, regional outbreaks ofdysentery. In recent years, Sd1 has caused epidemic dysentery in Central America, south Asia, andcentral and southern Africa. An epidemic in Central America from 1969 to 1973 was responsible formore than 500,000 cases and 20,000 deaths. The epidemic in central and southern Africa began in 1979and has affected at least nine countries. It is likely that most developing countries are at risk of epidemicdysentery due to Sd1.
These guidelines are intended to assist national health authorities, public health officers and health careproviders in their efforts to prevent and/or treat Sd1 disease. The text describes the epidemiology,clinical features and management of disease caused by Sd1, and interventions that can reduce both theincidence of Sd1 infections and mortality due to Sd1 disease. ABOUT SHIGELLA DYSENTERIAE TYPE 1 (SD1) Shigella are the most important cause of acute bloody diarrhoea (dysentery). Shigella cause dysenteryby invading and destroying cells that line the large intestine, leading to mucosal ulceration, ahaemorrhagic inflammatory exudate and bloody diarrhoea. Apart from bloody stools, patients withdysentery often have fever, abdominal cramps and rectal pain. In almost half of cases, however,Shigella cause acute non-bloody diarrhoea that cannot be distinguished clinically from diarrhoea causedby other enteric pathogens.
Sd1 differs from the other Shigella serogroups (S. flexneri, S. sonnei, and S. boydii) in three importantways: (i) only Sd1 causes large and prolonged epidemics of dysentery, (ii) antimicrobial resistanceoccurs more frequently among Sd1 than other Shigella serogroups, and (iii) infection with Sd1 causesmore severe, more prolonged, and more frequently fatal illness than does infection with other Shigellaserogroups.
Sd1 disease is most often severe or fatal in young children, especially infants, and in the elderly and themalnourished. Although most patients recover without complications within seven days, persistentdiarrhoea may occur occasionally. Other complications of Sd1 infection are haemolytic-uraemicsyndrome (HUS), seizures, sepsis, rectal prolapse and toxic megacolon. The case-fatality rate withoutprompt effective treatment ranges from 1% to 10%.
Infection with Sd1 is most common in overcrowded areas with poor sanitation, sub-standard hygiene,and unsafe water supplies. Refugee populations may be at especially high risk. During epidemics, up toone-third of the population at risk may be infected. Illness tends be seasonal, occurring during hot, wetweather. Seasonality is less pronounced, however, in Africa. Transmission of Sd1 probably occursmostly through person-to-person contact and through contaminated food and water. The infectious doseis low; the ingestion of as few as 10-100 organisms has caused disease in volunteers. During dysentery, Shigella are excreted in large numbers in the stool (106-108 bacteria per gram). Shigella have been documented to survive in soiled linen for up to seven weeks, in fresh water for 5-11days, in salt water for 12-30 hours, in dust at room temperature for six weeks, in sour milk for four weeks and in kitchen refuse for 1-4 days. Survival is prolonged at temperatures below 25oC. Freezingwill not eliminate the organism, although it may reduce the number that survive. OTHER CAUSES OF DYSENTERY
Aside from Sd1 and other Shigella, endemic dysentery is caused by Campylobacter jejuni,enteroinvasive Escherichia coli, Salmonella and, infrequently, Entamoeba histolytica.
Enterhaemorrhagic E. coli O157:H7 has caused localized outbreaks of dysentery in Europe and NorthAmerica, usually associated with eating undercooked contaminated beef or drinking raw milk; personto person transmission also occurs. Between 5% and 10% of patients with severe bloody diarrhoeadevelop HUS. Approximately 20% of persons with HUS die and an additional 30% develop chronicrenal failure. A related organism, E. coli O157:NM, has caused at least one large outbreak of dysenteryin southern Africa. Laboratory techniques for identifying E. coli O157 are given in Annex 9. E. histolytica is an occasional cause of dysentery, especially in young adults, but does not causeepidemic disease. Asymptomatic infection with E. histolytica is, however, frequent in developingcountries, being present in up to 10% of healthy persons. In some epidemics of dysentery due to Sd1, E. histolytica was identified and initially thought to be the cause. Because of this incorrect diagnosis,persons with dysentery were treated with anti-amoebic drugs (such as metronidazole), with resultingcontinued transmission of Sd1 and excess preventable mortality. Finding cysts of E. histolytica inbloody stool during an epidemic does not indicate that it is the cause of the epidemic, or even that it isthe cause of dysentery in an individual patient. PREVENTION OF INFECTION WITH SHIGELLA DYSENTERIAE
Infection with Sd1 is spread by direct contact with an infected person, or by eating or drinkingcontaminated food or water. Preventive measures are summarized below. Health education
Health education is the key to public awareness and cooperation. Experienced health educators play animportant role in epidemic control. Community groups and service organizations can also assist bydisseminating messages through their programmes.
The public must be informed about how infection by Shigella is spread and how it can be prevented. Messages should be spread through home visits, health facilities, schools, religious leaders and the massmedia. Messages must be carefully prepared, taking into consideration the local terminology, culturalsensitivities, traditions and beliefs. Only measures that it is possible to implement and that have a highlikelihood of preventing transmission of the disease should be promoted. Particular attention should begiven to the strategies presented below, which are also effective for reducing morbidity and mortalityfrom endemic shigellosis and from acute watery diarrhoea caused by other pathogens, such as cholera. Some examples of public health messages are contained in Annex 1. Hand-washing with soap
Hand-washing with soap may be the most effective measure to prevent transmission of Shigella; itshould be promoted in every family. Hand-washing is particularly important after defecation, aftercleaning a child who has defecated, after disposing of a child's stool, before preparing or handling foodand before eating.
Hand-washing is practised more frequently where water is plentiful and within easy reach. If possible,water for washing should be stored separately from drinking-water. During an epidemic of Sd1 disease,soap should be provided to those without it. If soap is not available, ash or earth can be used to scrubthe hands. Washed hands should not be dried with dirty cloths.
Breastfeeding
Breastfeeding of infants and young children should be promoted. Infants and children who are breastfedhave fewer episodes of diarrhoea or dysentery due to Shigella; when these do occur, they are less severethan in those who are not breastfed. This protection is greatest in infants who are exclusively breastfeduntil 4-6 months of age, but remains significant when breastmilk is given with other foods, even into thethird year of life. Food safety
Each country should establish adequate controls for the handling and processing of food through anational programme on food safety. Environmental health workers should monitor food-handlingpractices and be given the authority to stop street sales or close restaurants when their inspections revealunsanitary practices.
Health education for the general population should stress the following messages concerning thepreparation of food for adults, children and infants (see also Annex 2):
do not eat raw food, except undamaged fruits and vegetables that are peeled and eatenimmediately;
eat food while it is still hot, or reheat it thoroughly before eating;
wash and thoroughly dry all cooking and serving utensils after use;
keep cooked food and clean utensils separate from uncooked foods and potentiallycontaminated utensils;
wash hands thoroughly with soap before preparing food;
protect food from flies by means of fly screens. Safe drinking-water
An adequate quantity of safe water must be available for drinking. Water supplies should be adequateto meet all the needs of a population all year round. It is recommended that a minimum of 20 litres ofwater per person per day be available. Health clinics and hospitals require 40-60 litres per patient perday. Ideally, no dwelling should be located more than 150 metres from a water source.1,2 Generalguidelines for ensuring a safe water supply are given below. United Nations High Commissioner for Refugees Handbook for Emergencies. Geneva, 1982.
Cairncross S, Feachem RG. Environmental health engineering in the tropics: an introductory text. New York,John Wiley and Sons Ltd., 1983:28-33. 4.5.1 Water supply
Piped water must be properly chlorinated. Recommended chlorine levels for piped water are given inAnnex 3. Leaking joints should be repaired and constant pressure should be maintained in the system toprevent the entry of contaminated groundwater.
Where an exposed water source (a river, pond, or open well) is used for drinking-water, it should beprotected from contamination by people and animals. This may require that a fence be built around it. Drainage ditches should be dug to prevent storm water and other surface water from flowing into thedrinking-water source. Defecation must not be allowed within 10 metres of the water source, andshould be downhill, or downstream, from it. Wells should be equipped with a well-head drainage apron,and with a pulley, windlass, or pump. Other water sources should be provided for bathing, washing andother purposes.
Where locally available water is likely to be contaminated, drinking-water should be supplied by tankersor transported in drums, provided it is adequately chlorinated and a regular supply can be ensured. Thetrucking of water is, however, expensive and difficult to sustain; it is usually considered a short-termmeasure until a local supply can be established. 4.5.2 Home storage and treatment
Families should be encouraged to store drinking-water in covered containers that are cleaned daily, tokeep drinking-water away from children and animals, and to use a long-handled dipper, kept speciallyfor the purpose, to take water from the containers. Another approach is to store drinking-water in anarrow-mouthed container, with an opening too small to allow the insertion of a hand. When the safetyof drinking-water is uncertain, it should be chlorinated in the home (see Annex 3) or boiled. Heatingwater until it starts to boil vigorously is adequate to kill Shigella and other bacterial pathogens. Boiledwater should be stored in a separate sealed or covered container. Water used for purposes other thandrinking need not be boiled. Disposing of human excreta
High priority should be given to ensuring the safe disposal of human waste. Sanitary systemsappropriate for local conditions should be constructed with the cooperation of the community. Designsfor latrine construction in different types of soils and climatic conditions can be found elsewhere.3 Seealso Annex 4 for instructions on making a ventilated improved pit latrine.
Health education messages should stress the need for proper use of latrines by everyone, includingchildren. They should also stress the dangers of defecating on the ground or in, or near, the watersupply. The disposal of children's excreta in latrines should be emphasized. If children defecate on theground, the faeces should be picked up, using a scoop or shovel, and deposited in a latrine or buried.
When large groups of people congregate, as for fairs, funerals or religious festivals, particular care mustbe taken to ensure the safe disposal of human waste. Where there is no latrine, defecation should beperformed in marked areas and a shovel provided to bury the faeces.
Franceys R, Pickford J, Reed R. A guide to the development of on-site sanitation. Geneva, World HealthOrganization, 1992 (ISBN 92 4 154443 0). Preventing spread of Shigella dysenteriae type 1 in health facilities
The following steps can help to reduce the spread of Sd1 infection in clinics and hospitals:
provide plenty of water and soap for hand-washing, preferably in easily accessible, highlyvisible locations;
wash hands with soap before and after examining each patient;
ensure that health workers who care for dysentery patients (or other diarrhoea patients) do notprepare or serve food;
dispose of stools of dysentery patients in a latrine or toilet (if this is not possible, bury them);
wash and disinfect the clothes and bed linen of dysentery patients frequently. Disinfecting clothing and disposing of bodies
Prompt and thorough disinfection of a patient's clothing, personal articles and immediate environmentcan help to control spread of infection within a family. Effective and inexpensive disinfectants include: chlorinated lime powder, 2% chlorine solution, and a 1-2% solution of phenol. Clothes should bewashed thoroughly with soap and water, and then boiled or soaked in disinfectant solution. Sun-dryingof clothes is also helpful since direct sunlight will kill Sd1. Utensils may be washed with boiling wateror disinfectant solution. The washing of contaminated articles, particularly clothes, in rivers and pondswhich might be sources of drinking-water, or near wells, must be prohibited.
Funerals of persons who die with diarrhoea, whether bloody or not, should be held quickly and close tothe place of death. The washing of dead bodies and the preparation and distribution of food duringfunerals should be discouraged. These should never be done by the same persons. Antimicrobial prophylaxis
Giving an antimicrobial to prevent transmission of Sd1 dysentery is never indicated. It has not beenshown to be effective and it can hasten the emergence of resistant strains, making treatment of thedisease more difficult.
PREPARATION FOR SHIGELLA DYSENTERIAE TYPE 1 EPIDEMICS
A strong national Control of Diarrhoeal Disease (CDD) Programme is the best long-term preparation foran epidemic of dysentery caused by Sd1. Countries with well-established national CDD programmeshave effective disease surveillance systems, trained health professionals, reliably supplied healthfacilities, and ongoing health education activities. Programmes in various government ministries anddepartments work together to improve water supply, sanitation and food safety practices.
When an outbreak of dysentery occurs in the area or nearby, these activities need to be reinforced andapplied to its control. If these activities are not yet established, they must be developed urgently. Specific preparatory measures are described below. Coordinating committee
An interministerial committee should be developed to plan and coordinate the response to outbreaks ofcommunicable diseases, including dysentery. The committee should include the manager of the nationalCDD programme. It may also be appropriate to establish similar committees at subnational levels. Thecommittee's objective should be to ensure rapid implementation of effective control measures. Somespecific functions of the committee should be to:
make a comprehensive plan of preparation for an epidemic;
coordinate the efforts of various governmental sectors;
collaborate with regional and international organizations;
collect and report information on dysentery cases and deaths;
procure, store and distribute essential supplies;
implement, supervise, monitor and evaluate control activities.
In the event that no such committee exists when an outbreak threatens, one should be created.
Surveillance and reporting
For surveillance and reporting purposes, the case-definition of dysentery is diarrhoea with visible bloodin the stool. To detect outbreaks of dysentery, treatment facilities should record systematically andreview regularly all cases of bloody diarrhoea. Records should include the name, age, date of visit andaddress for each patient, the clinical diagnosis, and the treatment given. Ideally, these data should besummarized and reported weekly to facilitate early detection of epidemics. A dysentery outbreak shouldbe suspected whenever there is an unusual increase in the weekly number of patients with bloodydiarrhoea or deaths from bloody diarrhoea.
When an outbreak of dysentery is detected, local and provincial (or national) health authorities shouldbe notified immediately. Reports should specify the number of patients, their ages, the dates of onset ofillness, and the names of towns or villages affected. Bacteriological studies should be done promptly todetermine whether Sd1 is the cause (see Annex 7). The national CDD Programme manager or theEpidemic Control Unit of the Ministry of Health should be informed immediately of all bacteriologicresults, so that appropriate control measures can be started. Reports of outbreaks should also be sharedwith neighbouring countries, as dysentery epidemics do not respect national borders. Althoughinternational notification of epidemic dysentery is not required, the local WHO representative and otherappropriate authorities should be informed. Laboratory
The overall role of the laboratory is described in section 8. Some steps that should be taken to preparefor a possible outbreak of Sd1 infection are as follows:
Prepare at least one laboratory for isolation of Shigella. Not every laboratory needs to becapable of isolating Shigella. It is preferable to have one well-equipped laboratory with suitablytrained staff to which specimens can be quickly and safely transported, than to have several thatare equipped and staffed inadequately. Obtain appropriate transport media. See Annex 5. Make provision for cold transport of stool specimens. Specimens to be cultured for Shigellashould be transported promptly to the laboratory at 4°C. See Annex 5. Have the necessary supplies at the designated laboratory. See Annex 6.
When an outbreak is reported the urgent need is to identify the causative organism and determine itsantimicrobial susceptibility. Procedures for collection of stool specimens, identification of Sd1 and drugsusceptibility testing are in Annexes 5, 7 and 8. Treatment policy
The mainstay of treatment for Sd1 disease is appropriate antimicrobial therapy, which lessens the risk ofserious complications and death. Other supportive measures used in treatment of acute diarrhoea shouldalso be provided.
A national treatment policy for epidemic dysentery caused by Sd1 should be prepared that includes:
giving an antimicrobial effective for Sd1;
giving ORS solution or other fluids to prevent or treat dehydration;
providing follow-up and referral for persons at increased risk of serious morbidity or death. 5.4.1 Selection of effective antimicrobials
The selection of recommended antimicrobials should be based on recent susceptibility testing of Sd1strains from a nearby area or, after an epidemic develops, obtained locally. Guidelines for susceptibilitytesting are in Annex 8. Antimicrobials that should be considered are listed in Table 1. Antimicrobialsshould be selected that are:
effective against at least 80% of local (or nearby) Sd1 strains;4
available locally or rapidly obtainable.
Unfortunately, resistance of Sd1 to ampicillin and cotrimoxazole has become widespread. Nalidixicacid, formerly used as a "backup" drug to treat resistant shigellosis, is now the drug of choice in mostareas, but resistance to it is also appearing. The fluoroquinolones and pivmecillinam (amdinocillinpivoxil) are still effective for most strains of Sd1, but most are costly and may not be readily available. When the presence of Sd1 has not been confirmed, or its antimicrobial susceptibility is not yet known,nalidixic acid should be selected until more precise information is available.
Antimicrobials that are not effective for Sd1 are listed in Table 2. These include: (i) agents to which Sd1strains are usually resistant, and (ii) those to which Sd1 strains are sensitive in vitro, but which penetratepoorly the intestinal mucosa where invasive Sd1 must be killed. These agents, and antimicrobials to
If the best available antimicrobial has lower efficacy, e.g. 50%, it should be used until a more effective onecan be obtained.
which Sd1 strains are resistant in vitro, should not be selected. 5.4.2 Use of antimicrobials when the supply is limited When the supply of an effective antimicrobial is not sufficient to treat all cases, priority for treatmentshould be given to those at highest risk of death, as described in section 7.2. Vigorous efforts shouldalso be made to obtain a sufficient stock of an effective antimicrobial for treatment of all cases ofbloody diarrhoea. This may require importing an antimicrobial that is not available locally.
Summary of antimicrobials for treatment of infections with Shigella dysenteriae
a Low = <US$ 1.00; Medium = US$ 1-4.00; High = US$ 5-30.00. Cost will vary from place to place and over time. Costs presented are for five days' treatment for an adult when antimicrobials are purchased in large quantities. b All antimicrobials should be given for five days. c To determine a child's dose, multiply the dose/kg by the child's weight. However, the child's dose should never be more than that shown for adults. d New quinolones have not yet been approved for use in children below 12 years of age. There is growing evidence, however, that they will prove both safe and effective. They are already used by some workers to treat children with serious illness caused by strains of Sd1 resistant to all other available agents. Antimicrobials that are not effective against Shigella dysenteriae type 1 1. Antimicrobials to which strains of Shigella are usually resistant in vitro: 2. Antimicrobials to which Shigella may be sensitive in vitro, but with no documented efficacy in vivo:
Nitrofurans (e.g. nitrofurantoin, furazolidone)
Aminoglycosides (e.g. gentamicin, kanamycin)
First- and second-generation cephalosporins (e.g. cephalexin, cefamandole)
Emergency stocks of essential supplies
Health facilities must have access to adequate quantities of essential supplies, including: appropriateantimicrobials, oral rehydration salts (ORS) and intravenous fluids. During a dysentery epidemic thesesupplies may be needed quickly and in greater quantities than usual.
Sufficient stocks should be maintained at appropriate points in the drug delivery system. Small reservestocks should be kept at local health facilities, larger buffer stocks at district or provincial sites, and anemergency stock at a central distribution point. Buffer stocks should be sufficient to meet a suddenincrease in demand for specific supplies. Buffer and emergency stocks should be rotated regularlythrough the normal delivery system to avoid their becoming outdated.
A system is required to monitor the use of buffer and emergency stocks and to ensure their promptreplacement. The need for emergency supplies should be determined and individuals assigned tocoordinate their procurement and distribution. The national coordinating committee should beresponsible for procuring supplies and equipment from external agencies, ensuring that all drugs andmaterials are appropriate and avoiding duplication of requests (see section 5.1). A single central systemfor recording all incoming supplies and their distribution within the country is advised.
The supplies and equipment needed to manage 100 cases of dysentery are listed in Annex 10. Training in case management
Medical and paramedical personnel should receive intensive and continuing training to ensure they arefamiliar with the most effective techniques for managing patients with acute diarrhoea, includingdysentery. WHO can provide materials for clinical management training that emphasize hands-onpractice in assessing and treating patients with diarrhoea.5,6 These are appropriate for training healthworkers in dysentery case management as part of preparedness for epidemics. Mobile control teams
Where peripheral health services are not prepared for epidemic dysentery, or are overwhelmed by it,mobile teams may be formed to:
collect stool specimens for submission to a bacteriology laboratory;
establish and operate temporary treatment centres;
provide on-the-spot training in case management;
supervise environmental sanitation and disinfection activities;
carry out health education activities for the community;
provide the emergency logistical support, such as delivery of essential supplies.
The teams may consist of doctors, nurses, paramedical staff, health educators and technicians. Briefingof team members on their duties, and any required training, should be done during preparation for adysentery epidemic or when one is first detected. PRINCIPAL STEPS IN THE MANAGEMENT OF PATIENTS WITH DYSENTERY CAUSED BY SHIGELLA DYSENTERIAE TYPE 1
Effective treatment of patients with bloody diarrhoea during an Sd1 epidemic consists of the followingsteps (see also Figure 1):
Refer immediately to hospital persons who are severely malnourished, appear seriously ill or arein another high-risk category.
Treat all cases promptly with an oral antimicrobial effective against local Sd1 strains.
Treat and prevent dehydration with oral rehydration therapy, or intravenous (IV) therapy ifseverely dehydrated.
Give frequent small meals of the patient's usual food; continue to breastfeed infants and youngchildren. Diarrhoea management training course: guidelines for conducting clinical training courses at health centresand small hospitals. Geneva, World Health Organization, 1990 (WHO document CDD/SER/90.2). The management and prevention of diarrhoea; practical guidelines, 3rd ed. Geneva, World HealthOrganization, 1993 (ISBN 92 4 154454 6). DETAILS OF MANAGEMENT OF PATIENTS WITH DYSENTERY CAUSED BY SHIGELLA DYSENTERIAE TYPE 1
Public health messages should encourage all persons who develop bloody diarrhoea to reportimmediately to the nearest health facility with appropriately trained and supplied health workers. Community health workers on home visits should also help to find and refer cases for treatment. Thetreatment strategy is described below. Diagnosing dysentery
The diagnosis of dysentery is made by observing blood in a fresh stool specimen or by asking thepatient, or the mother of a child, whether the stools are bloody. These methods usually have equalsensitivity and precision. If there is doubt that a history of bloody stool is accurate, observation of afreshly passed stool is essential. 7.2 Identification of high-risk patients
Individuals at increased risk of death from dysentery caused by Sd1 are:
children less than 5 years of age (infants, severely malnourished children7 and children whohave had measles in the past six weeks are at highest risk);
anyone who is dehydrated, has had a convulsion or is seriously ill when first seen;
older children and adults who are obviously malnourished. Referral to hospital
Children with severe malnutrition (weight-for-age less than 60% or weight-for-length less than 70% ofNational Center for Health Statistics medians) and any patient who is seriously ill should be referredimmediately to hospital.
Other high-risk patients should also be referred to hospital, if space is available. Otherwise they shouldbe treated as outpatients with careful follow-up to ensure they are improving in response toantimicrobial therapy. 7.4 Antimicrobial therapy
Appropriate treatment requires an oral antimicrobial that is effective against local strains of Sd1 (seeTable 1). If possible, one should be selected that is effective against all Sd1 strains. If an effectiveantimicrobial is unavailable or in limited supply, treatment guidelines must be revised. Both situationsare considered below.
This refers to children with weight-for-age <60%, or weight-for-length <70%, of National Center for HealthStatistics median values, oedema of both legs, or mid-arm circumference less than 12.5cm (red band of astandard tape). 7.4.1 When an effective oral antimicrobial is available
Treat patients for five days. Give outpatients enough antimicrobial to last five days and instruct thepatient (or mother) how to take it.
When an effective antimicrobial is taken, clinical improvement (i.e. feels better, fewer stools, less bloodin the stool, less fever, less abdominal pain, improved appetite) normally occurs within 48 hours. Suchimprovement provides reassurance that the patient, although not yet fully recovered, is respondingsatisfactorily to treatment.
All high-risk patients treated as outpatients should be reviewed after two days of treatment. Any whoare not improving, as defined above, should be admitted to hospital. Other outpatients who return afterat least two days of treatment and have not improved should also be admitted to hospital.
If the antimicrobial these patients received was not effective against all local Sd1 strains and a secondeffective antimicrobial is available, the first one should be stopped and the second one given forfive days. If a second antimicrobial for Sd1 is not available, the first one should be continued forfive days. All patients should be given supportive care as described in section 7.5. 7.4.2 When an effective antimicrobial is in limited supply
Sometimes the supply of an effective antimicrobial is insufficient to treat all persons with dysentery. Insuch instances steps should be taken urgently to obtain a sufficient supply of an effective antimicrobial. Until this is achieved, the available supply of effective drug should be reserved for the high-risk patientsdescribed above and for patients whose illness worsens without antimicrobial therapy. Antimicrobialsthat are known to be ineffective or to which the local Sd1 strain is resistant should not be given . Allpatients, however, should receive the supportive treatment summarized below.
It should be emphasized that an effective antimicrobial should be given to all patients with dysenterycaused by Sd1. Patients who do not receive an effective antimicrobial, even though not diagnosed ashigh-risk, may still have a severe or fatal outcome of their illness. Supportive care
Optimal treatment of dysentery caused by Sd1 includes preventing or treating dehydration andcontinuing to feed, as described in WHO guidelines for the management of acute diarrhoea.8
7.5.1 Preventing and treating dehydration
Although dysentery is not usually associated with marked loss of fluid and electrolytes, the patient'sstate of hydration should be accurately assessed. If dehydration is detected, it should be treated at thehealth facility with ORS solution (for some dehydration) or IV fluids (for severe dehydration). Patientswith dysentery and signs of dehydration are at increased risk for complications and should be re-evaluated after two days of treatment. All patients should be encouraged to take increased amounts ofsuitable fluids at home, such as ORS solution, rice water, soup, yoghurt-based drinks and plain water. 7.5.2 Nutritional management
Continued provision of nutritious food is important for all patients with dysentery. However, owing to
The treatment of diarrhoea: a manual for physicians and other senior health workers. Geneva, World HealthOrganization, 1995 (WHO document WHO/CDR/95.3).
anorexia, patients may have to be coaxed to eat. Initially, food may be refused, but appetite usuallyimproves after 1-2 days of effective antimicrobial therapy. Frequent small meals with familiar foods areusually better tolerated than a few large meals.
Infants and young children should breastfeed as often and as long as they want. Infants below 4 monthswho already take solid foods should continue to receive them. Mothers of infants younger than 4months who are not exclusively breastfed should be advised and helped to establish exclusivebreastfeeding. Infants older than 4 months, and young children, should be offered their usual foods. Young children convalescing from dysentery should be given an extra meal each day for at least twoweeks to help them recover any weight lost during the illness. The caretakers of children with pre-existing malnutrition should be advised on appropriate feeding practices and the child monitored untilsubstantial weight gain has been documented. See Annex 11 for a summary of feeding practices duringand after diarrhoea.
Adults should eat easily-digestible, nutritious foods, avoiding those that are fried or spicy.
7.5.3 "Antidiarrhoeal" drugs
Drugs available for symptomatic relief of abdominal and rectal pain, or to reduce the frequency of stools(e.g. loperamide, diphenoxylate, paregoric) should never be used in the treatment of shigellosis as theymay cause severe adverse effects. Treatment of complications 7.6.1 Potassium depletion
Potassium depletion may be quite severe in shigellosis. It can be prevented by replacing faecal losseswith ORS solution. Giving potassium-rich foods, such as bananas or green coconut water, is alsohelpful.
7.6.2 High fever
High fever (more than 39°C) can cause seizures in young children. It should be controlled by givingparacetamol. Reducing fever also improves appetite and reduces irritability. 7.6.3 Haemolytic-uraemic syndrome
Haemolytic-uraemic syndrome (HUS) is an unusual but serious complication of dysentery that affectsthe blood clotting system and kidneys. It may follow infection with Sd1 or E. coli O157:H7. Theclassic triad of symptoms is haemolytic anaemia, thrombocytopenia and renal failure. HUS may bemild, with rapid recovery, or severe, with kidney failure. Haemodialysis may be required. Clottingabnormalities can cause bleeding, and the red blood cell count may be low. Transfusions of wholeblood or platelets are often needed in severe cases. With adequate treatment, many HUS patientsrecover fully.
HUS should be suspected when a dysentery patient develops easy bruising and has little or no urineoutput. The diagnosis of HUS can be made by the following: (i) a low haematocrit, (ii) a blood smearshowing fragmented red blood cells, (iii) a low platelet count, or platelets not seen on the blood smear,and (iv) elevated levels of blood urea nitrogen or serum creatinine. When this occurs, stop givingpotassium-rich foods or fluids, including ORS solution, and refer the patient to hospital. Role of the laboratory
The principal tasks of the laboratory are to:
isolate Sd1 when an epidemic is first reported;
determine the antimicrobial susceptibility of the Sd1 isolates to guide antimicrobial therapy;
monitor antimicrobial susceptibility of Sd1 isolates regularly during the epidemic to detect anyimportant changes.
After an epidemic caused by Sd1 has been confirmed, it is not necessary to examine specimens from allcases or contacts. In fact, this should be discouraged as it places an unnecessary burden on laboratoryfacilities and is not required for effective treatment.
The laboratory must inform government health officials, clinicians and epidemiologists promptly of allrelevant findings. National laboratories may obtain technical assistance from WHO or one of itsCollaborating Centres (Annex 12). Determine the cause of the epidemic
When a dysentery epidemic is first reported, stool specimens should be collected from 10-20 untreatedcases to determine if Sd1 is the cause. The method for collecting and transporting stools is described inAnnex 5, and the method for isolating and identifying Sd1 is described in Annex 7. A list of essentialsupplies is found in Annex 6. If Sd1 is not identified, stool specimens should be cultured for E. coliO157:H7 (see Annex 9).
If difficulty is encountered, WHO reference laboratories can assist national laboratories to isolate andidentify Shigella, including Sd1, from faecal samples. Specimens should be shipped by express air mail(see Annexes 5 and 12). Determine antimicrobial susceptibility of Shigella dysenteriae type
The technique for determining antimicrobial susceptibility of Sd1 is described in Annex 8. It isrecommended that initial antimicrobial susceptibilities be confirmed at a WHO reference laboratory.
The susceptibility of Sd1 to antimicrobials can change dramatically during an epidemic. It is important,therefore, that susceptibility be reassessed regularly, for example, every 2-6 months. For seasonalepidemics, susceptibility testing should also be performed at the end of the epidemic season to allowantibiotic policy for the following season to be determined. A plan should be established as part ofepidemic preparedness to collect 10-20 specimens from untreated patients in different affected areas andtransport them to the designated laboratory. The antimicrobial susceptibility of Sd1 isolates should bedetermined and any important changes reported promptly, so that necessary changes in recommendedantimicrobial treatment can be made. Reference laboratories
A national reference laboratory should be able to isolate and identify Shigella, including Sd1, andperform antimicrobial susceptibility testing, or at least have access to an international referencelaboratory with those capabilities. The reference laboratory should also be responsible for trainingregional and local health staff in appropriate isolation and transport techniques, monitoring the qualityof the laboratory services, and ensuring that the results of laboratory testing are disseminated to regional
AFTER AN OUTBREAK
Careful clinical surveillance should be continued to ensure that sporadic cases of shigellosis arepromptly detected and treated. Efforts to improve personal and domestic hygiene, water supplies andsanitation to help prevent a recurrence of the epidemic should also be continued.
Routine laboratory examinations of food and water are not likely to be helpful. The experience gainedduring the epidemic should be used to strengthen the capacity of the national CDD programme tocontrol all endemic acute diarrhoea, including shigellosis, and help prevent further epidemics. ACKNOWLEDGEMENTS
The following persons assisted in preparation and review of this document: Dr Serge Malé, UNHCR,Geneva, Switzerland; Dr John Murray, BASICS, Arlington, VA, USA; Dr Christophe Paquet,EPICENTRE, Paris, France; Dr Allen Ries, Centers for Disease Control and Prevention, Atlanta, GA,USA; Dr Ronald Waldman, BASICS, Arlington, VA, USA. Sample public health messages for prevention of Shigella dysenteriae infection
The following sample messages are valid for all forms of acute diarrhoea, including dysenteryand cholera. They should be adapted to local conditions and translated into local languages:
THREE SIMPLE RULES FOR PREVENTING DYSENTERY
1. Cook your food2. Boil or chlorinate your drinking-water3. Wash your hands
ARE YOU PROTECTED FROM DYSENTERY? DO YOU PREPARE FOOD SAFELY? Cooking kills dysentery germs
Thoroughly cook all meats, fish and vegetables. Washing protects from dysentery
Wash your hands before preparing or serving food.
Wash your dishes and utensils with soap and water.
Wash your cutting board especially well with soap
Peeling protects from dysentery
Eat only fruits that have been freshly peeled, such as
KEEP IT CLEAN: COOK IT, PEEL IT, OR LEAVE IT!
ARE YOU PROTECTED FROM DYSENTERY? IS YOUR DRINKING-WATER BOILED OR TREATED?
Even if it looks clean, water can contain dysentery germs.
Water for drinking can be made safe in two ways:
Boil it to kill dysentery germs. Chlorine kills dysentery germs: use three
drops of chlorine solution for each litre of water, mix well, and leave itfor half an hour before drinking. To make the chlorine solution: mix three level tablespoons (33 grams) of bleaching
This quantity is for a bleaching powder that contains 30% concentration by weight
of available chlorine. The quantity to be recommended must be adapted for the bleachavailable on the local market. ARE YOU PROTECTED FROM DYSENTERY? IS YOUR DRINKING-WATER STORED SAFELY?
Clean water can become contaminated again if it is not stored safely.
Store drinking-water in a clean container with a small opening or acover. Use it within 24 hours.
Pour from the water container - do not dip a cup into the container.
KEEP IT CLEAN: STORE DRINKING-WATER SAFELY
ARE YOU PROTECTED FROM DYSENTERY? DO YOU WASH YOUR HANDS?
The germs that cause dysentery are invisible. They can be carried on your hands
Always wash your hands:
after you use the toilet or latrine, or clean up your children;
before you eat or feed your children. What is the best way to wash your hands?
Wash all parts of your hands - front, back, between the
ARE YOU PROTECTED FROM DYSENTERY? DO YOU USE A TOILET OR LATRINE?
Dysentery germs live in faeces. Even a person who is healthy might have dysentery
Always use a toilet or latrine. If you don't have one - build
Keep the toilet or latrine clean.
Dispose of babies' faeces in the toilet or latrine (or
Wash your hands with soap (or ash) and clean water
Rules for preparing food safely to prevent dysentery9 1. Cook foods thoroughly
Foods can easily become contaminated with the germs that causes dysentery. Thoroughcooking will kill the germs, but remember that all parts of the food must become hot. Do noteat uncooked foods, unless you peel or shell them yourself.
2. Eat cooked foods immediately
When cooked foods cool to room temperature, bacteria can begin to grow. To be on the safeside, eat cooked foods as soon as they come off the heat. When there is a delay betweencooking and eating food, as when food is sold in restaurants or by street vendors, it should bekept over heat, at 60°C or more, until it is served. 3. Store cooked foods carefully
If foods must be prepared in advance or kept as leftovers, be sure to store them in a refrigeratoror ice box below 10°C or in a hot box kept continuously above 60°C. Otherwise, cooked foods
that have been stored for more than two hours must be thoroughly reheated before being eaten. 4. Reheat cooked foods thoroughly
Reheating foods thoroughly before eating is your best protection against germs that may havegrown during storage. Thorough reheating means that all parts of the food must become hot. Eat food while it is still hot. 5. Avoid contact between raw foods and cooked foods
Safely cooked food can become contaminated, and potentially dangerous, through even theslightest contact with raw food. Contact can be direct, as when raw fish comes into contact withcooked foods. It can also be indirect, as when a cutting surface and knife are used to prepareraw fish and then, without washing, to slice cooked food. Doing so can reintroduce all thepotential risks of illness that were present before cooking. 6. Wash hands frequently
Wash hands thoroughly before you start preparing food and after every interruption, especially ifyou have to "change" or clean up the baby or have used the toilet or latrine.
7. Keep all kitchen surfaces clean
Any surface used to prepare food must be kept absolutely clean. Think of every food scrap,crumb or spot as a potential source of germs. Cloths used for washing or drying foodpreparation surfaces, dishes and utensils should be changed every day and boiled before reuse.
Adapted from Annex 6, Golden rules for safe food preparation, in Health surveillance and managementprocedures for food-handling personnel: report of a WHO consultation. Geneva, World Health Organization,1989 (WHO Technical Report Series, No.785). 8. Use safe water
Safe water should be used when preparing food that will not be cooked or when making ice fordrinks. If there are any doubts about the water, boil it or treat it with chlorine before it is used. Making water safe by chlorination The following guidelines should be translated into messages that take into account locally-available products and measuring devices, and whether the instructions are for home orinstitutional use. Make a stock solution of chlorine (1% concentration by weight of available chlorine). Add to 1 litre of water: Product (% concentrationby weight of available chlorine)
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
Bleaching powder orchlorinated lime (30%)
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
If products with these concentrations of chlorine are not available, adjust the amount usedaccording to the available concentrations.
Store the stock solution in a cool place in a closed container that does not admit light. The stock solution must be used no later than one month after it is made. Use the stock solution to make water safe. Add water to stock solution to ensure complete mixing:
Allow the chlorinated water to stand for at least 30 minutes before using it. The residualchlorine level after 30 minutes should be between 0.2 and 0.5 mg/litre.
If the water is turbid (not clear, with a lot of suspended solid matter):
n boil it vigorously for 1 minute, instead of treating it by chlorination. Recommended chlorine levels in water distribution systems in areas affected by epidemic dysentery
The minimum levels of free residual chlorine necessary for safe water are:
at all sampling points in a piped water system. 0.5 mg/litre
Regular monitoring is required to ensure that these minimum levels of chlorine aremaintained. Building a ventilated improved pit latrine10
A ventilated improved pit latrine is a practical means of disposing of human excreta and may bea good solution for use in rural areas. The decision on the type of latrine to be selected shouldtake account of local factors such as type of soil and density of population.
The latrine must be constructed at least 30 metres from wells or other sources of drinking-waterand, where possible, at least six metres from houses. It should not be located uphill from thewater source or dug in marshy soil.
A latrine 2 metres deep with an opening 1 metre x 1 metre can be used by a family of fivefor 2-4 years. (This assumes an accumulation rate of between 60 and 100 litres per person peryear.) To keep bad odours and flies to a minimum, ventilation can be provided by an externalvertical vent, topped by a fly screen. The edges of the pit should be raised above ground level toprevent rain or other water from draining into it. The latrine should have a concrete or woodenslab that reaches the walls of the superstructure. Where possible, concrete reinforced with steelwires at least 8 mm in diameter and 150 mm apart should be used because of its durability andresistance.
The slabs and floor should be washed daily and disinfected regularly with cresol or bleachingpowder. After the pit is loaded to two-thirds of its capacity (1.3 metres height), it should befilled with soil and compacted, and a new pit should be dug.
10 For more specific instructions, see Cairncross S. Small scale sanitation. London, London School of Hygiene
Collection and transport of stool specimens for Shigella
Specimens that cannot be cultured within one hour of collection should be placed in a transportmedium and refrigerated immediately. Unlike some organisms, Shigella will die, even intransport media, if they are not refrigerated.
Selection of transport media
Cary-Blair transport medium is a semi-solid medium useful for the preservation and transport ofspecimens for Shigella, as well as Escherichia coli, Salmonella, Vibrio cholerae, Vibrioparahaemolyticus, and Yersinia enterocolitica. It is stable when stored in tightly sealedcontainers. It can be used for 18 months or longer under proper conditions of storage, providedthere is no loss of volume and no evidence of contamination or colour change. Other transportmedia that are similar to Cary-Blair are Amies' and Stuart's transport media.
Buffered glycerol saline (BGS) is also useful for transporting specimens for Shigella as well asEscherichia coli and Salmonella. BGS is considered to be better than Cary-Blair for Shigella,provided that the BGS is still alkaline, as indicated by the pink colour that persists after theaddition of faeces. BGS is unsuitable for Vibrio or Campylobacter. Its disadvantages are that itis a liquid medium and that it can be used for only one month after it is made.
Selection of cases for bacteriologic sampling
When an outbreak of dysentery occurs, laboratory analysis of a small number of adequatelycollected clinical specimens is sufficient to provide the diagnosis. The key is to collect thespecimens properly and to transport them rapidly to a fully equipped clinical laboratory. Thisapproach permits rapid diagnosis of outbreaks at low cost. 10-20 cases should be selected forsampling at each investigation site. Cases should meet all of the following criteria:
onset of illness less than four days before sampling;
have not received antibiotic treatment for this illness;
Collection of specimens
Two swabs of rectal contents or of fresh stool (less than one hour old) should be collected fromeach case selected. If possible, the Cary-Blair transport media should be refrigerated for an hourbefore use so the swabs can be placed into cool medium. Insert swab into the Cary-Blair first inorder to moisten it, then insert it 1-1½ inches into the rectum and rotate it gently. Remove theswab and examine it to ensure that the cotton tip is stained with faeces. Insert the swabimmediately into the tube of transport media. Push the swab to the bottom of the tube ofmedium. Repeat with the second swab, placing it in the same tube as the first swab. Break offthe top part of the sticks. Tighten the screw top firmly.
Labelling of specimens
Use the attached Stool Specimen Data Sheet to record information on each case. Assignnumbers to collected specimens in consecutive order. Always write the numbers on the frostedportion of the specimen tube using an indelible marker pen. If no frosted area is present, applyfirmly a piece of first aid tape and write on it.
Transport of specimens
Refrigeration of specimens after collection is essential. If the specimens will arrive at the laboratorywithin two days, they can be refrigerated at 4°C. Pathogens can still be recovered from refrigeratedsamples up to seven days after collection, although the yield decreases after the first two days. Refrigeration during transport can be achieved for up to 36 hours by shipping in a well insulated boxwith frozen refrigerant packs or wet ice.
If it is impossible for specimens to reach a laboratory within two days, they can be frozen,although this will decrease the number of organisms present and the likelihood of isolating thepathogen. They should be frozen as soon as possible after collection and held at -20°C. Freezing at conventional freezer temperatures (-5° to 0°C) is not acceptable, as it allowsthawing and refreezing, which will quickly reduce the number of organisms present. Frozenspecimens should be shipped with dry ice, observing the following precautions:
protect the specimens from direct contact with dry ice, as intense cold can crack theglass tubes;
protect the specimens from carbon dioxide by sealing the screwcaps with electrical tapeor by sealing the tubes in a plastic bag;
ensure that the container is at least one-third full of dry ice. If specimens are shipped byair and more than 2 kg of dry ice is used, special arrangements may be necessary withthe air carrier.
The shipping arrangements should be determined before specimens are collected. Within-country shipping may be by ground or by air. To ship longer distances (e.g. to a referencelaboratory or WHO Collaborating Centre) overnight express air mail is ideal. As wet ice in thebox will not last more than 36 hours, arrangements should be made for immediate pickup at thereceiving airport. When shipped, communicate the following information immediately to thereceiving laboratory: the air bill number, the flight number, and the times and dates of departureand arrival of the flight. Address the package clearly, including the name and telephone numberof the receiving laboratory. Write in large letters: EMERGENCY MEDICAL SPECIMENS;CALL ADDRESSEE ON ARRIVAL; HOLD REFRIGERATED. Contents of transport kit
1 roll of first aid tape (for labelling tubes)
Cary-Blair medium Preparation: Dissolve the ingredients in the water while heating in a boiling water-bath, stirring until the solution is clear (do not allow it to boil). After cooling to 50°C, add 9 ml of freshly prepared 1% calcium chloride solution and adjust the pH to 8.4 (with N/10 sodium hydroxide). Dispense 7 ml amounts into cleaned and sterilized 9 ml screw-capped bottles (e.g. Bijou bottles), leaving a small air space at the top and the caps loosened. Sterilize by steaming at 100°C or in a boiling water-bath for 15 minutes, and tighten the caps after sterilization. Record the batch date on the label and store in a cool dark place. STOOL SPECIMEN DATA SHEET BACTERIOLOGIC SURVEILLANCE
* If antibiotics were taken, list type of antibiotic, dose and number of days taken
Name __________________________________________
Title ___________________________________________
Name __________________________________________
Address ________________________________________
________________________________________________
Phone/Fax/Telex _________________________________
Supplies needed for laboratory identification of Shigella dysenteriae (for 100 cases) S. dysenteriae (group A)S. flexneri (group B)S. boydii (group C)S. sonnei (group D)
TMP/ SMXNalidixic acidPivmecillinamCiprofloxacin (or otherfluoroquinolone)
Control strains (susceptible andresistant)
Laboratory identification of Shigella11 Enrichment
No enrichment medium is suitable for Shigella.Preparation of faecal suspension
Suspend faeces from rectal or faecal swab in a tube containing 1 ml of saline (0.85% NaCl). Wash the swab thoroughly in the saline by swirling the tube, and rotate the swab against the sideof the tube to express the fluid from the swab.
Portions of formed stools should also be suspended in saline to make a turbid suspension. Liquid stools require no addition of saline.
The plate can be inoculated with the faecal suspension, or directly from the swab, and thenstreaked with a loop.
Direct inoculation of agar plates
Use a moderate inoculum (2 or 3 loopfuls of faecal suspension). Incubate plates at 35-37°Cfor 18-24 hours.
Inoculate a general purpose plating medium of low selectivity and one of moderate or highselectivity. MacConkey agar is recommended as a medium of low selectivity. MacConkey agarwith 1 mcg/ml of potassium tellurite has been reported to be particularly useful for S. dysenteriae type 1 (Sd1). Use a small inoculum. Incubate at 35-37°C for 18-24 hours.
Xylose-lysine-desoxycholate (XLD) agar is recommended as a medium of moderate or highselectivity for isolation of Shigella. Desoxycholate citrate agar (DCA) is a suitable alternative.
Do not use salmonella-shigella (SS) agar, as it often inhibits growth of Sd1. Each new batch of medium should be controlled for quality before routine use by inoculating itwith known reference strains and observing their growth and colony characteristics. Identification of colonies on plating media
Colonies suspicious for Shigella will appear as follows:
MacConkey agar: Convex, colourless, 2-3 mm
11 For additional information see: Manual for laboratory investigations of acute enteric infections. Geneva,
World Health Organization, 1987 (WHO document CDD/83.3 Rev. 1).
XLD agar: Red, smooth, 1-2 mmDCA agar: Colourless, translucent, 2-3 mm
Identify well separated colonies of typical appearance to be transferred from each of the platingmedia for further testing by making a mark on the bottom of the Petri plate.
Whenever possible a person experienced with the identification of Shigella should trainlaboratory workers who are not familiar with its identification.
Inoculation of Kligler iron agar (KIA)
Pick three characteristic colonies from the plating media and inoculate into KIA as follows: stab the butt and then streak the slant with a zig-zag configuration. Pay attention to properlabelling of the tubes. If screw-cap KIA tubes are used, make sure that the caps are loose. Incubate overnight. On the following morning, examine the reactions in the KIA tubes. Tubessuspicious for Shigella will have an acid (yellow) butt and an alkaline (red) slant. They will notproduce gas (no bubbles or cracks in the agar) and will not produce hydrogen sulfide (no blackalong the stab line).
Triple sugar iron agar (TSI) can also be used for the identification of Shigella. It will give thesame reactions as KIA.
Serological tests of cultures suspected of being Shigella
Agglutination tests are carried out on a clean glass slide. Use a straight wire to remove a portionof the growth from the surface of the KIA slant and emulsify in a 3 mm loopful of physiologicalsaline. Mix thoroughly by tilting back and forth for about 30 seconds and then examinecarefully to ensure that the suspension is smooth and does not show clumping due to auto-agglutination. If clumping occurs, the culture is rough and cannot be serotyped. If thesuspension is smooth (turbid and free-flowing), add one loopful of antiserum, mix well usingthe loop, and observe for agglutination over a period of 60 seconds against a dark background. If the reaction is positive, clumping will appear within 30 seconds to one minute. Interpret theagglutination tests as shown below:
If agglutination occurs with group A, report:
Test with S. dysenteriae type 1 antiserum. If positive, report:
If agglutination occurs with group B, report:
If agglutination occurs with group C, report:
If agglutination occurs with group D, report:
For further details on identifying Shigella and other Enterobacteriaceae, refer to the WHOManual for laboratory investigations of acute enteric infections, CDD/83.3 Rev.1 (1987). Media preparation
MacConkey agar, desoxycholate agar, and Kligler's iron agar are commercially available as pre-mixed powders. Preparation of media from individual ingredients is described below. MacConkey agar (modified)
It may also be prepared with meat extract broth as indicated below:
Add solutions 1 and 2, as described below
Preparation of base medium: Add progressively and dissolve lactose, bile salts, peptone, and NaCl in one litre of meat extract broth while heating at approximately 80°C in a water-bath and stirring. Dissolve the agar by heating the broth in a boiling water-bath. Adjust the pH to 7.2-7.4 with 0.1 N sodium hydroxide. Distribute in 200-ml amounts in screw-capped bottles with caps loosened; sterilize by autoclaving at 121°C for 15 minutes. Tighten the caps after sterilization. Record the batch date on the label and store. Preparation of Solution 1: Dissolve 1 g of neutral red in distilled water; make up to a volume of 100 ml. Heat the solution in steam at 100°C for 30 minutes. Label and store in a cool area or at 4°C. Preparation of Solution 2: Dissolve 0.1 g of crystal violet in distilled water; make up to a volume of 100 ml. Heat the solution in steam at 100°C for 30 minutes. Label and store in a cool area or at 4°C. Pouring plates: Melt 200 ml of the base solution in a boiling water-bath. Cool to about 60°C. Add aseptically 0.6 ml of solution 1 and 0.4 ml of solution 2. Mix well and pour into 90 mm sterile Petri dishes. Sterility test: Incubate the plates at 37°C for 24 hours and examine for contamination. Performance test: Prepare an 18-hour broth culture from the stock cultures of S. typhi, E. coli, and S. flexneri.
Mix the broth cultures of S. typhi and E. coli in a 1:10 ratio (volume to volume). Prepare a 106 dilution of the mixture (take one 4 mm loopful of the mixture and add it to10 ml of sterile physiological saline, mix well, then take a 4 mm loopful of the dilutionand add to another 10 ml of sterile saline).
Prepare a 106 dilution of the S. flexneri broth culture in a similar manner.
Inoculate the plating media with 4-mm loopfuls of the dilute mixtures of S. typhi, E. coliand S. flexneri.
Incubate the plates at 37°C overnight and examine for growth of typical colonies. Xylose lysine desoxycholate agar (XLD agar) Basal medium: Directions for complete medium: Add all ingredients to the water and heat to boiling to obtain a complete solution. Cool to 50-55°C, adjust reaction so that pH after sterilization will be 6.9, and autoclave at 121°C for 15 minutes. Cool to 50-55°C and add aseptically the following solutions in the amount indicated:
20 ml of thiosulfate-citrate solution (to prepare, dissolve 34 g of sodiumthiosulfate and 4 g of ferric ammonium citrate in 100 ml of water; sterilize byfiltration);
25 ml of a 10% aqueous solution of sodium desoxycholate (sterilize by filtration or byautoclaving at 121°C for 15 minutes).
Mix well, readjust pH if necessary to 6.9, and pour in 15-20 ml amounts into Petri dishes. Desoxycholate citrate agar (DCA) (modified) Base medium: Solution 1: Solution 2: Preparation of base medium: Adjust the reaction of the broth to pH 8.0-8.4 and dissolve the agar by heating in a boiling water-bath or in steam at 100°C. Filter the molten agar immediately upon removal from heating through multilayer surgical gauze. Adjust to pH 7.4; add 2.5 ml of freshly prepared 1% solution of neutral red and 10 g of lactose and 10 g of proteose peptone. Mix well and distribute in 200-ml amounts in screw-capped bottles. Sterilize by heating in steam at 100°C for one hour followed by autoclaving at 110°C for 10 minutes. Tighten the caps, record batch date on the label and store at 4°C. Preparation of plates: Melt 200 ml of the base medium and cool to about 80°C. Add aseptically 10 ml of solution 1 and the appropriate volume of solution 2 (as indicated in the following procedure on titration) using two different pipettes; mix well after each dilution. Distribute into sterile Petri dishes. The medium must cool rapidly, otherwise it may become too soft. Record the batch number and date on the label. Titration of sodium desoxycholate: Melt seven bottles of the base medium and label from 6 to 12. Add 10 ml of solution 1 to each bottle. Add respectively 6, 7, 8, 9, 10, 11, and 12 ml of solution 2 to the bottles 6, 7, 8, 9, 10, 11, and 12. Mix well and pour plates (label plates with the same numbers as the bottles). Select the plates that give the best growth of Salmonella and Shigella. Record the volume of solution 2 used. Use: This medium is selective for Salmonella and Shigella. Salmonella will produce raised colourless or translucent colonies. Shigella will produce opaque ground-glass colonies. It must be remembered that other non-lactose-fermenting organisms will grow on DCA, and these need to be differentiated from Salmonella and Shigella by biochemical tests. Lactose-fermenting organisms form raised colonies often surrounded by a red halo. Kligler's iron agar (KIA)
Ferrous sulfate (FeSO . Preparation: Dissolve the agar in the meat infusion broth (see below), or alternatively in meat extract broth, by heating in a boiling water-bath or in steam at 100°C. Bring the molten nutrient agar to 80°C in a water-bath. Add and dissolve the lactose, peptone, proteose peptone, NaCl, glucose, ferrous sulfate, and sodium thiosulfate and mix well. Adjust the pH to 7.4. Add 6 ml of 0.5% solution of phenol red and mix well. Distribute in screw-cap tubes (15 x 150 or 16 x 160 mm) in 5-6 ml amounts and sterilize by autoclaving at 121°C for 15 minutes. Allow the medium to cool and set with a slant of 2.5 cm and a butt 2.5 cm deep. Record batch number and date on the label and store at room temperature not exceeding 25°C.
For preparation of meat infusion broth and meat extract broth, see below. Alternatively, anadditional 10 g of peptone, 3 g of beef extract, 3 g of yeast extract, and one litre of distilledwater may be used in place of meat infusion broth. Sterility test: Incubate the tubes at 37°C for 18-24 hours and examine for contamination. Performance test: Inoculate five tubes with the following cultures: S. typhi, S. paratyphi B, E. coli, Citrobacter freundii and Shigella sonnei. Incubate at 37°C for 18-24 hours and examine for correct reactions. Preparation of meat extract broth
Put 500 g of lean minced meat (beef heart or fat-free meat) into a pan or casserole and add onelitre of water; place it in the refrigerator (4°C) overnight. Next morning, bring to a boil andsimmer for 15 minutes, while stirring with a glass rod. Filter through a wet paper filter toremove fat. Add water to make one litre (to replace that lost in boiling). Preparation of meat infusion
Several media contain an infusion from, for example, beef heart or veal heart. Heat one litre of1/20 N aqueous sodium hydroxide to boiling and add 1000 g of minced fat-free fresh meat ororgan. Mix thoroughly, bring to a boil, and simmer for 20 minutes stirring frequently. Thereaction of the mixture should be about pH 7.5. Strain through several layers of cheesecloth,squeeze out excess liquid, adjust the volume to 1000 ml with distilled water, and use the liquidimmediately for making up media as specified in the formulations.
Add 450 ml of the infusion to 550 ml of distilled water, then add other ingredients. Dispense in5 ml volumes in tubes, and sterilize by autoclaving at 121°C for 15 minutes. Adjust final pH to7.4. Antimicrobial susceptibility testing of Shigella12 Types of susceptibility tests
The dilution test: For quantitative estimates of antimicrobial activity, dilutions of theantimicrobial may be incorporated into broth or agar medium, which is then inoculated with thetest organism. The lowest concentration that prevents visible growth after overnight incubationis known as the minimum inhibitory concentration (MIC) of the agent.
The diffusion test: Paper discs or tablets impregnated with the antimicrobial are placed on agarmedium uniformly seeded with the test organism. A concentration gradient of the antimicrobialis formed by diffusion from the disc and the growth of the test organism is inhibited at adistance from the disc that is related, among other factors, to the susceptibility of the organism.
There is an approximate linear relation between the log MIC as measured by a dilution test andthe inhibition zone diameter obtained in the diffusion test. A regression line expressing thisrelation can be obtained by testing a large number of strains by the two methods in parallel, andshould be established when locally prepared discs are used.
The interpretation of the diffusion test results should always be based on this correlationbetween MIC and inhibition zone, combined with information on the therapeutically obtainableconcentrations of the antimicrobials.
The procedure for the diffusion test is described below.
Agar: Use one of the media recommended by the manufacturer of the discs. This will allowyou to interpret inhibition zones using guidelines recommended by the manufacturer.
If the recommended media are not easily obtainable, one of the commercially available media(e.g. Mueller-Hinton medium, DST agar, Sensitest agar, Iso-sensitest agar, Wellcotest,Sulphonamide-Antagonist-Free medium) may be tested to see whether there are majordifferences in the inhibition zones as compared with the recommended media.
If a new medium is to be used, it should be checked to determine:
whether the inhibition zone of the control strains (see table) is correct;
if possible, whether the concentration of divalent ions is correct.
The medium should be poured in Petri dishes with an agar depth of 4 mm or more (25 ml in a
12 For additional information see: Manual for laboratory investigations of acute enteric infections. Geneva, World
Health Organization, 1987 (WHO document CDD/83.3 Rev. 1).
9 cm plate). Dry the plates before use. Store unused plates for not more than two weeks in therefrigerator, preferably in a sealed plastic bag. Take care that they do not become too dry. Table: Quality control - susceptibility of control strains
Nutrient broth: Any nutrient broth that is available in the laboratory can be used, e.g. trypticasesoy broth (for preparation, see below.) The broth should be distributed in 3-5 ml quantities andsterilized by autoclaving.
To prepare, dissolve the ingredients in the water, dispense, and autoclave at 121°C for 15
Inoculum for the indirect susceptibility test
The indirect susceptibility test should be used for faecal bacteria. This requires that Shigella areinoculated from a pure culture. For most disc methods semiconfluent growth is recommended,and it is essential to use a standardized inoculum in order to perform a reliable susceptibilitytest. Use the inoculation method recommended by the manufacturer of the discs or tablets youare using. One of the following is usually recommended:
Make a turbidity standard by adding 0.5 ml of 1.175% (w/v) barium chloridedihydrate (BaCl .
2 2H2O) solution to 99.5 ml of 1% sulphuric acid. The turbidity
standard solution should be placed in a tube identical to the one used for thebroth sample. It can be stored in the dark at room temperature (22-25°C) for six
months, provided it is sealed to prevent evaporation.
Touch 5-10 colonies of similar appearance with a loop and transfer to a tube ofbroth, or transfer a loopful of confluent growth of a pure culture to a tube ofbroth.
After incubation at 35°C for 4-6 hours, compare the broth culture with the
turbidity standard. This comparison can be made more easily if the tubes areviewed against a background of white paper with print of various sizes on it. Adjust by diluting the broth culture with sterile broth or saline.
Inoculate the plates by dipping a sterile cotton swab into the inoculum. Removeexcess inoculum by pressing and rotating the swab firmly against the side of thetube above the level of the liquid. Streak the swab all over the surface of themedium three times, rotating the plate through an angle of 60° after each
application. Finally, apply the swab all round the edge of the agar surface. Leave the inoculum to dry for a few minutes at room temperature with the lidclosed.
This method of inoculation should give nearly confluent growth.
If broth is used, overnight cultures of enterobacteria should be diluted about 10-4(1:10000).
When plates are used, prepare the suspension as follows: dip a 0.01-ml loop intofive colonies, transfer to 1 ml saline, dip another 0.01-ml loop in this suspension,transfer to 1 ml saline, and finally dip a third 0.01-ml loop in this suspension andtransfer to 5 ml saline. Mix the suspension well. Flood the plates.
This method of inoculation should give semiconfluent growth. Choice of antimicrobial
Only a limited and carefully selected number of antimicrobials should be included in thesusceptibility test. The most appropriate ones are: ampicillin, TMP-SMX, nalidixic acid,pivmecillinam and ciprofloxacin (or another fluoroquinolone).
Any commercially available discs or tablets with the proper potency can be used. The discpotency recommended in WHO guidelines is shown in the table. Avoid using discs withdifferent potencies, as these can give misleading results.
Discs prepared locally: If antimicrobial discs cannot be obtained commercially, they can beprepared locally. Buy or cut 6-mm filter paper discs pretested for absence of antibacterialproperty. Place them separately on the bottom of a sterile Petri dish. Pipet 20 µl ofantimicrobial solution on each disc. Use an antimicrobial solution that will give the discpotency shown in the table. Dry the disc for about one hour at 35°C. The paper discs may also
be placed on the inoculated agar surface prior to pipetting the appropriate antimicrobialsolutions.
Storage: Stocks of antimicrobial discs should preferably be kept at -20°C; the freezer
compartment of a home refrigerator is convenient. A small working supply of discs can be keptin the refrigerator for up to one month. On removal from the refrigerator, the containers shouldbe left unopened at room temperature for about one hour. This procedure reduces the amount ofcondensation that occurs when warm air enters the cold container. If a disc-dispensingapparatus is used, it should have a tight-fitting cover and be stored in the refrigerator. It shouldalso be allowed to warm to room temperature before opening. Antimicrobial tablets (exceptcarbenicillin) are stable for at least four years at room temperature.
Application of antimicrobials: The antimicrobial discs or tablets may be placed on theinoculated plates in zones properly divided and marked on the back, using either a pair of sterileforceps, an antimicrobial disc dispenser, or a sterile needle tip.
Not more than seven discs (one in the centre, six 15 mm from the edge of the plate) can beplaced on a plate.
Incubation: The plates should be placed in an incubator at 35°C within 30 minutes of their
preparation. Do not incubate in an atmosphere of carbon dioxide. Do not stack more than two
Measurement of inhibition zones
After overnight incubation the diameter of each inhibition zone (including the diameter of thedisc) is measured and recorded in mm. The measurements can be made with a ruler on theunder-surface of the plate without opening the lid. If the medium is opaque, the zone can bemeasured by means of a pair of callipers.
The end-point of inhibition is judged by the naked eye at the edge of the clear zone wheregrowth starts. With sulfonamides and sulfamethoxazole-trimethoprim, however, slight growthoccurs within the inhibition zone; such growth should be ignored. Interpretation of zone sizes
The result of the susceptibility test, as reported to the clinician, classifies the microorganism inone of three categories:
Susceptible (S): A microbe is called "susceptible" to a drug when the infection causedby it is likely to respond to treatment with this drug, at the usual dosage.
Intermediate (I): The susceptibility of an organism is called "intermediate" when theinfection is likely to respond to unusually high doses of the drug, or when the organismis located in a part of the body where the drug is concentrated (urine, bile, intestinallumen, local application). The intermediate category also acts as a buffer zone andcompensates for small technical errors in the performance of the procedure.
Resistant (R): This term implies that the infection is not likely to respond to a givenantimicrobial, irrespective of the dosage and of the location of the infection.
The translation of the zone of inhibition into a susceptibility category depends on whether anabsolute or a comparative method has been used.
Absolute method: This is performed with as high a degree of standardization as possible(medium, inoculum, etc.). This implies that for each antimicrobial one inhibition zone alwayscorresponds to one MIC-value. Moreover, by means of a regression line and fixed MIC-breakpoints between S, I, and R, it is possible to translate the inhibition zone of the organismtested into S, I, or R according to the recommendations of the manufacturer.
Comparative method: This is based on a comparison of the inhibition zone of an unknownstrain with that of a control strain and is less satisfactory than the absolute method. The controlstrains should be included every day and treated exactly as the test strains using identical mediaand inoculum density (semiconfluent growth). The control strains and the test strains can evenbe inoculated on the same plate on each side of the same disc, thus providing a control for everydisc. This method is especially recommended if the performance of the disc is very poor andvariable. E. coli NCTC 10418 and Staph. aureus NCTC 6571 are recommended as controlstrains in the comparative method. If the inhibition zones are measured from the edge of thedisc to the edge of the zone, the following interpretation is used:
Susceptible: Zone size equal to, wider than, or not more than 3 mm smaller than thecontrol.
Intermediate: Zone size greater than 3 mm, but smaller than the control by more than 3mm. Day-to-day controls
All susceptibility tests are very sensitive to small variations in media, inoculum, incubation,temperature, etc. In order to perform a reliable test it is of the utmost importance every day toinclude control strains in the test.
If absolute methods are used, the following control strains should be included daily: Staph. aureus (ATCC 25923) and E. coli (ATCC 25922).
In comparative methods, control strains are automatically included every day in order tointerpret the inhibition zones of the unknown strains. Records should be kept of the inhibitionzones of the control strains to detect large variations. A decrease in potency of the discs onstorage may be revealed by a decrease in the size of the inhibition zone around a control strain.
The control strains for both the absolute and comparative tests can be obtained from theAmerican Type Culture Collection13 or other national culture collections. They are provided inthe form of pellets of desiccated pure cultures. Cultures for day-to-day use should be grown onslants of nutrient agar (trypticase soy agar is convenient) and stored in the refrigerator. Theyshould be subcultured onto fresh slants every two weeks. The control strains are treated just likethe other pure cultures investigated by the susceptibility test, and the inhibition zones recorded.
When the procedure is correctly performed, the zone sizes shown by the control organismsshould fall within the range of diameters given in the table. The limits that can be tolerated inthe test have been determined in a collaborative study involving a large number of reputablelaboratories, and reflect the degree of accuracy than can be routinely obtained by a good clinicallaboratory. When results fall regularly outside this range, they should be regarded as evidencethat one or more technical errors have been introduced into the test or that the reagents are atfault. Each reagent, and each step in the test, must be investigated until the cause of the errorhas been eliminated.
Grossly aberrant results that cannot be explained by technical errors in the procedure mayindicate contamination or sudden changes in the susceptibility or growth characteristics of thecontrol strain. If this occurs, a fresh control strain should be obtained from a reliable source.
1. American Type Culture Collection (ATCC), 12301 Parkland Drive, Rockville, MD 20852, USA2. National Collection of Type Cultures (NCTC), Central Public Health Laboratory, Colindale Avenue,
Isolation and identification of Escherichia coli O157 Escherichia coli O157 is not detected by the usual methods used to isolate and identifytraditional enteric bacterial pathogens. However, the isolation and identification of E. coli O157can be performed by most laboratories, given the proper medium and antiserum.
Background E. coli O157 ferment D-sorbitol slowly or not at all, unlike most other E. coli strains whichferment sorbitol rapidly. This finding led to the development of sorbitol-MacConkey agar,which is now commercially available. Sorbitol-negative colonies are colourless and areconsidered "suspicious for E. coli O157". Other E. coli colonies are pink to red.
Preparation of faecal suspension: See guidelines in Annex 7.
Plating on sorbitol-MacConkey agar: Each new batch of medium should be controlled forquality prior to routine use by inoculating it with known reference strains and testing for growthand colony characteristics. Streak one loopful of suspension onto the plate. Incubate 24 hoursat 36°C. Sorbitol-negative colonies are colourless and are considered "suspicious for E. coliO157".
Agglutination: Strains of E. coli O157 rapidly agglutinate in O157 antiserum, which iscommercially available. Select three isolated sorbitol-negative colonies for agglutination. Follow the manufacturer's instruction for the specific agglutination procedure. Once a positivecolony is encountered, additional colonies need not be tested, and the specimen is considered"positive for E. coli O157".
If sorbitol-MacConkey agar is not used, 5-10 lactose-positive colonies from MacConkey agar ora similar plating medium can be screened in tubes of sorbitol fermentation medium. At leastfive colonies should be selected for screening because the organism is not always present inpure culture. Colonies that do not ferment sorbitol within 24 hours are screened foragglutination in E. coli O157 antiserum, as outlined above.
Isolated strains should be sent to a reference laboratory for confirmation and further typing. Treatment supplies for 100 persons in dysentery epidemics Sanitary/hygienic supplies
200 grams of hand soap per person per month30 boxes soap for washing clothes2 one-litre bottles of cleaning solution (2% chlorine or 1-2% phenol)
Rehydration supplies
100 packets ORS (for one litre each)20 bags Ringer's lactate solution (one litre each)5 scalp vein sets10 adult IV giving sets
Antimicrobials For adults: 400 one-gram tablets of nalidixic acidFor children: 400 one-gram tablets of nalidixic acid
Other antimicrobials may need to be substituted for nalidixic acid depending on localantimicrobial susceptibility patterns.
Other treatment supplies
One large water dispenser5 one-litre bottles for ORS solution5 half-litre bottles for ORS solution10 tumblers5 teaspoons
Assumptions:
20% of cases are children <5 years. All are treated with antimicrobials.
80% of cases are >5 years old. All are treated with antimicrobials.
20% of cases will have dehydration requiring ORS.
10% of cases will have dehydration requiring IV fluids.
Each person will be given 200 grams of hand soap per month.
Each family will be given soap for washing clothes and bed linen of ill person. Feeding during and after diarrhoea General guidelines
Encourage the child to eat during the entire illness
During and after illness, feed the child as follows:
Breastfeed as often as the child wants, day and night.
For children taking other milk, give appropriate milk as often as the child wantsby cup. Increase the breastfeed while gradually reducing the other milk overseveral days. Give the additional milk by cup, not by bottle
If the child is fed on other milk alone, give this as often as the child wants bycup, not by bottle.
Give foods three times a day if breastfed or five times a day if not breastfed
Give family foods: three meals + two additional feeds. WHO collaborating centres for Shigella The following two centres may be approached for technicalassistance in bacteriological aspects of Shigella:
WHO Collaborating Centre for ShigellaDiarrheal Diseases Laboratory SectionFoodborne and Diarrheal Diseases BranchNational Center for Infectious DiseasesNational Center for Disease Control and Prevention (CDC)Atlanta, GA 30333USA
WHO Collaborating Centre for Phage-typing and Resistance of EnterobacteriaDivision of Enteric PathogensCentral Public Health LaboratoryColindale AvenueLondon NW9 5HTUnited Kingdom
Bibliography
Bennish ML, Harris JR, Bogdan JW, Struelens M. Death in shigellosis: incidence and riskfactors in hospitalized patients. Journal of Infectious Diseases, 1990, 161:500-506.
Bennish ML. Potentially lethal complications of shigellosis. Reviews of Infectious Diseases,1991, 13(Suppl 4):S319-24.
Bhattacharya SK, Bhattacharya MK, Dutta P, Sen D, Rasaily R, Moitra A, Pal S. Randomizedclinical trial of norfloxacin for shigellosis. American Journal of Tropical Medicine andHygiene, 1991, 45(6):683-687.
Butler T, Islam MR, Azad MAK, Jones PK. Risk factors for development of hemolytic uremicsyndrome during shigellosis. The Journal of Pediatrics, 1987, 10(6):894-897.
Griffin PM, Tauxe RV. The epidemiology of infections caused by Escherichia coli O157:H7,other enterohemorrhagic E. coli, and the associated hemolytic uremic syndrome. AmericanJournal of Epidemiology, 1991, 13:60-98.
Huppertz HI. An epidemic of bacillary dysentery in Western Rwanda, 1981-1982. CentralAfrican Journal of Medicine, 1986, 32:79-82.
Kabir I, Butler T, Khanam A. Comparative efficacies of single intravenous doses of ceftriaxoneand ampicillin for shigellosis in a placebo-controlled trial. Antimicrobial Agents andChemotherapy, 1986, 29(4):645-8.
Keusch GT. Shigella. In Farthing, ed. Enteric infections. Mechanisms, manifestations, andmanagement. London, Chapman and Hall, 1988:265-82.
Malengreau M, Molima-Kaba, Gillieaux M, De Feyter M, Kyele-Duibone, Mukolo-Ndjolo. Outbreak of Shigella dysentery in eastern Zaire, 1980-1982. Annales des Sociétés belges deMédecine tropicale, 1983, 63:59-67.
Mathan VI, Bhat P, Kapadia CR, Ponniah J, Baker SJ. Epidemic dysentery caused by the Shigabacillus in a southern Indian village. Journal of Diarrheal Disease Research, 1984, 2(1):27-32.
Mendizabal-Morris CA, Mata LJ, Gangarosa EJ, Guzman G. Epidemic Shiga-bacillusdysentery in Central America: Derivation of the epidemic and its progression in Guatemala,1968-69. American Journal of Tropical Medicine and Hygiene, 1971, 20(6):927-33.
Reller LB, Rivas EN, Masferrer R, Bloch M, Gangarosa EJ. Epidemic Shiga-bacillus dysenteryin Central America: Evolution of the outbreak in El Salvador, 1969-70. American Journal ofTropical Medicine and Hygiene, 1971, 20:934-940.
Ries AA, Wells JG, Olivola D, Ntakibirora M, Nyandwi S, Ntibakivayo M, Griffin P, Tauxe R. Shigella dysenteriae type 1 infections in Burundi: The end of the line for antibiotics. Journal ofInfectious Diseases, 1994, 169:1035-9.
Rogerie F, Ott D, Vandepitte J, Verbist L, Lemmens P, Habiyaremene I. Comparison ofnorfloxacin and nalidixic acid for treatment of dysentery caused by Shigella dysenteriae type 1in adults. Antimicrobial Agents and Chemotherapy, 1986, 29(5):883-6.
Salam MA, Bennish ML. Antimicrobial therapy for shigellosis. Reviews of InfectiousDiseases, 1991, 13(Suppl 4):S332-41.
Salam MA, Bennish ML. Therapy for shigellosis. I. Randomized, double-blind trial of nalidixicacid in childhood shigellosis. The Journal of Pediatrics, 1988, 113(5):901-7.
Taylor DN et al. Introduction and spread of multi-resistant Shigella dysenteriae I in Thailand. American Journal of Tropical Medicine and Hygiene, 1989, 40(1):77-85.
Chemische Widerstandsfähigkeits-Tabellen Besondere Hinweise Die Chemischen Widerstandsfähigkeits-Tabellensind wertvolle Hilfen bei der Planung von Kunst-stoffrohrleitungen. Die in den Tabellen enthaltenenAngaben sind Ergebnisse aus Versuchen und prak-tischen Erfahrungen mit den Omniplast Rohrpro-grammen aus PVC-U, PE-HD und PP. Sie sind nichtohne weiteres auf alle Betriebsverh
Thematic report on protected areas or areas where special measures need to be taken to conserve biological diversity Please provide the following details on the origin of this report. Contracting Party: National Focal Point Full name of the institution: National Commission for Wildlife Conservation and Development (NCWCD) Name and title of contact officer: Prof Abdulaz
WHO/CDR/95.4