Metodología de los estudios caso-control en la epidemiología de la tuberculososis multirresistente

INTRODUCTION

Tuberculosis (TB) is the largest cause of death worldwide due a single infectious agent. It has been estimated to be responsible for around 7% of all deaths and 26% of preventible deaths throughout the world. Most of these deaths are among young adults. The emergence of the HIV epidemic in the last 10 years has not only increased the problem of TB, but has also raised questions over the adequacy of current diagnostic, treatment and prevention methods. Furthermore, the increase in multidrug-resistant TB, with high death rates especially among HIV-infected patients, has revealed the shortcomings in current control and prevention methods (1).

MYCOBACTERIAL RESISTANCE

From a bacteriological point of view, a population of Mycobacterium tuberculosis is resistant if 1% or more of the microorganisms multiply and develop in the presence of a specific concentration of a drug. Drug-resistant TB occurs when the number of bacilli resistant to the drug exceeds that of the bacilli susceptible to it. This occurs because of the selection and multiplication of resistant mutants which arises from inappropriate treatment. From a clinical point of view, there are two types of resistance to drugs: primary resistance and acquired (or secondary) resistance. Primary resistance is found in people who have never been treated for TB and who are infected with resistant microorganisms. Acquired resistance develops during treatment for TB, either because the patient has been treated with an inadequate regimen or because he or she did not correctly follow the regimen prescribed.

Multidrug-resistant TB is defined as the in vitro resistance of a strain of M. tuberculosis to two or more antituberculous drugs. From the clinical perspective, resistance to isoniazid and rifampicin is the most important pattern of resistance as it has been linked to a much lower cure rate than in drug-susceptible TB (2).

The World Health Organization (WHO) provides regular information on resistance to drugs for TB at a worldwide level. The overall trend is stable and even shows a slight decline if we look at initial resistance to one or more drugs. However, the increase in initial resistance to rifampicin and multidrug resistance in some countries are a cause for concern (3).

With regard to acquired resistance, which indicates that earlier treatment has failed, the available global reports show that this type of resistance has decreased over the last 30 years (3). The two main reasons for the development of clinical drug resistance are the lack of compliance with prescribed treatment and the use of inadequate treatment regimens.

The emergence of drug-resistant bacilli can be prevented by treating TB with a combination of two or more drugs. During the early phase of treatment, combined therapy is extremely important as this is when the bacterial population is at its highest (4). Antituberculous drugs vary in their ability to prevent the appearance of resistance to other drugs; the most effective seem to be isoniazid and rifampicin. In order to prevent the emergence of resistance when other comparatively weaker drugs are used, a combination of three drugs is required. Later, during continued treatment, combined therapy is of lesser importance because the level of the bacillary population is lower. Resistance to drugs is much less likely to occur in most types of extrapulmonary TB, which have much smaller populations of bacilli.

Drug resistance occurs more frequently in people living in parts of the world where the prevalence of drug-resistant TB is high (Southeast Asia, Latin America, Haiti and the Philippines). A number of studies have found high rates of primary drug resistance among these people, suggesting that drug-resistant TB is transmitted in the country of origin. Nevertheless, some patients treated previously may be mistakenly classified as cases of primary resistance, since it can be difficult to obtain an accurate history of their treatment.

Multidrug-resistant TB was reported shortly after the introduction of antituberculous drugs, but it was not until 1970 that outbreaks of this kind of TB were seen to be important. Between 1970 and 1990, there were microepidemics of TB resistant to isoniazid, as well as to several drugs; they affected a few people with close contact who had had prolonged or repeated exposure to probands.

From 1990 to October 1992, the Centers for Disease Control and Prevention (CDC) investigated epidemic outbreaks of TB resistant to numerous drugs in hospitals in Florida, New York and New Jersey, and in the penitentiary system in the state of New York.

In recent epidemics, the transmission has been from patient to patient, and from patient to health-care worker. Nosocomial transmission tests were confirmed by data on the haplospecific configuration of the bacteria's DNA: strains in cases linked from the epidemiological point of view had identical patterns according to the restriction fragment length polymorphism (RFLP) technique. There is no evidence that people infected with HIV are more likely to be infected by the TB bacillus if they are exposed to it. However, once infected, patients who have HIV infection are at a much greater risk of developing active disease than people who are not. In addition, TB infection may progress rapidly towards active disease. In these outbreaks, cases of active TB developed several weeks after exposure to the disease. These patients themselves went on to become a source of transmission, meaning that in a short space of time they caused multiple cases.

Prolonged infectiousness also aids transmission. The diagnosis of TB was delayed in people infected with HIV due to radiography patterns not typical of TB, coinfection with other pulmonary pathogens to which patients' symptoms were attributed, and to the excessive isolation in the laboratory of mycobacteria other than M. tuberculosis. Slow diagnosis led to delays in starting treatment and in taking steps to control the source of infection. In addition, resistance to antituberculous drugs was reported late, making effective treatment difficult and prolonging infectiousness.

One important risk factor in drug resistance is prior treatment with antituberculous medication. In a study conducted in New York by Frieden et al., the most reliable predictor of the presence of drug-resistant microorganisms was a history of previous treatment (5). Other studies have shown that drug-resistance rates increase with the duration of prior treatment. In most cases, drug resistance linked to prior treatment occurs because the treatment was inadequate (6). Furthermore, previous treatment has not been linked with drug resistance when short treatment regimens have been adequately supervised (7). Bacterial relapses after completion of supervised short-course chemotherapy with regimens containing both isoniazid and rifampicin are usually due to microorganisms susceptible to the drugs. Such patients respond well to a repeat of the previous treatment (8).

Another risk factor in multidrug-resistant TB is being in contact with a person who has infectious drug-resistant TB (9). Recent nosocomial outbreaks show a clear link between prior exposure to a patient who has infectious TB resistant to numerous drugs and the subsequent development of TB which is also resistant to the same drugs (10). Moreover, people previously treated for TB susceptible to antituberculous drugs may be reinfected with strains resistant to these drugs (11).

PATHOGENESIS, RISK FACTORS,CASES AND CONTROLS

Descriptive epidemiology studies do not explain the causes of diseases: they provide us with ideas of how they spread. In order to determine whether certain characteristics of a person, time and place give rise to health problems, we require analytical studies based on numerous comparisons. Analytical studies form the basis of etiological research and are useful in helping us determine the risk factors in multidrug-resistant TB.

According to the general definition of cause, a "cause" (etiological factor) must be modifiable, and its modification must produce changes in its effect (patients' state and propagation of the disease).

The causes of disease are often called risk factors. This term is frequently misused as it represents only part of the risk characteristics (i.e., any variable that may be associated with a high probability of disease). Grundy (12) perceives two elements in risk characteristics: risk markers (age, gender, etc.) which cannot be changed, but which nevertheless mean there is a high probability of disease in the future; and risk factors which are modifiable (smoking, diet, etc.).

In statistical terms, in keeping with the cause and effect sequence, epidemiology studies explore the relationships between independent variables (factors or risk markers, causes, etc.) and dependent variables (consequences, diseases, etc.). Identifying direct causes remains one of the major challenges in etiological research in medicine.

Etiological constellations or clusters of causes may appear at a given moment or in a particular sequence. If more than one "cause" is required to produce an effect, the "minimum sufficient causes" must be known. For example, an epidemic of multidrug-resistant TB is not due solely to the presence of isoniazid and rifampicin-resistant M. tuberculosis in the environment, but to inappropriate treatment, prolonged infectiousness, etc.

The most commonly used studies in epidemiology to determine the causes of multidrug-resistant TB are case-control studies. A case-control study may be defined as the comparison of earlier frequency of exposure (to a disease risk factor, protection factor or attribute) in at least two groups of subjects selected on the basis of their status with respect to a particular disease or condition. People with the disease (cases) and people without the disease (controls) are studied and compared in relation to attributes or exposure in the present or past that may be relevant to the development of the condition or disease (13). Instead of numerous measurements of the state of health and exposure, as required in cohort studies (at least two – one at the start and one at the end of a defined period), case-control studies are based on a single identification of the state of health and exposure in the past or present for each individual being studied. In terms of direction, a case-control study begins at the end of the natural history of the disease and "looks back" towards the exposure that might have given rise to the consequence (disease) being studied.

Generally, fewer individuals are analyzed than in a cohort study. A limited number of cases is usually defined and a control group is chosen that excludes cases (one or more controls per case). Exposure or lack of exposure is determined for all participants, and estimates of the strength and specificity of the causal link are obtained, while also saving time and resources.

DESIGN OF A CASE-CONTROL STUDY

Fundamental principles

It is not possible to calculate individual risks for exposed and unexposed subjects in a case-control study. Unfortunately, these studies are almost always based on prevalence (not incidence) and the denominators are ambiguous or unknown. Nevertheless, most of the studies of this type applied to multiresistance work with the number of incidences of tuberculosis reported in a period of time.

The best case-control study is one which has correctly neutralized systematic bias or errors (14); these errors may be derived from the selection of the cases and controls, from the gathering of information and the skewing that may be caused by confounding factors. Wacholder et al. (15) indicate four basic principles to be kept in mind when designing a case-control study: the basis of the study – to reduce bias in selection; the comparability of the measurement of exposure – to reduce bias in the information; "deconfounding" – to control the effect of other confounding factors; and efficiency – related to the appropriate use of time and resources.

Sample, case and control sources

One of the first problems that emerges when designing a case-control study is defining the population. Both the criteria and the source for selecting the subjects in the study must be fully representative of this base population. There are various sources that can be used for selecting cases and controls, including base population records, such as tumor records, records of epidemiological vigilance systems or vital statistics (deaths), and records from hospitals, primary health care centers, companies, insurance companies, etc.

Including individuals randomly selected from population records prevents bias in the selection and makes it possible to calculate the risk attributable to factors being studied in the population. The drawbacks are that this usually takes longer, it is more expensive to locate, identify and obtain information (16), and individuals selected in this manner are less likely to cooperate.Defining and selecting cases.

Defining and selecting cases

The study population may include all the cases that occurred in a particular period of time, or a sample may be selected when the number of cases is very large. Cases of TB that develop multiresistance within a period of time are selected for multidrug-resistant TB studies (17-19). Perfectly valid case-control studies can also be done by restricting cases by age, gender, place of residence or any other condition or characteristic of interest (20).

Suppose that we wish to conduct a case-control study to verify the association between the risk of multidrug-resistant TB and noncompliance with treatment. The cases could include all those of multidrug-resistant TB that occurred in a sample in a given year, or we could restrict it to cases in a district within the city, to those that occurred in penitentiary institutions, etc. Some authors (13) have advocated a case-restriction criterion based on the principle of the possibility of exposure: to restrict the study only to certain cases of multidrug-resistant TB (20-22).

The aim of this restriction would be to prevent a weakening of the estimated association and a decrease in the accuracy of the effect. In reality, however, this restriction criterion is related to efficiency: it reduces the number of subjects and interviews and reduces the cost of the study without affecting its validity, weakening the association or distorting the value of the estimated effect (23).

One important aspect regarding the determination of cases to be included in the study is the condition of the patient. It is possible to include only incident cases (i.e., recently diagnosed cases) or prevalent cases (cases already existing at the start of the study; 24), living cases or dead cases. The best approach is to take only incident cases and living subjects at the time of the interview. It should be kept in mind that incident cases have a better recollection of their past experience as it is more recent. Their survival is not affected by risk factors, as may occur in prevalent cases, and it is less likely that the disease will modify the exposure being studied. Prevalent cases may be included in the study in certain special situations, when there are not enough cases, and above all when dealing with uncommon diseases. It may be justifiable to include dead cases in the study in special circumstances, depending on the aims of the study and the sources of information used. When collecting detailed information on a subject's past, it should be remembered that if the source of information is the family, they will not know the details as well as the subject him or herself (25). If, however, the subject's clinical history or company records are used as a source of information, it is of no great importance whether the person is alive or dead (26).

It is also important to consider the criteria for defining cases. Published case-control studies on multiresistance establish clinical criteria (20) and bacteriological criteria that include the identification of the microorganism responsible for the process, tests on susceptibility to antimicrobials (27, 28) and even genetic constitution (18, 29). The diagnostic criteria used will affect whether or not false-positive cases are included in the study. The more false-positive cases are included in an etiological study on risk factors, the more the estimated association will be weakened. In the case of multidrug-resistant TB, the cases would be TB patients with a strain of M. tuberculosis resistant to at least isoniazid and rifampicin, although clinical and/or therapeutic criteria that classify the multiresistance as primary or acquired may be added (28).

Definition and selection of controls

Selecting controls has been seen as one of the most difficult issues in a case-control study. The most appropriate controls must be carefully defined for each study. The idea that controls must be similar to cases in all aspects except for not having the disease is not correct (30).

The first requirement in selecting a control is that he or she must come from the same population as the case. A practical aspect to bear in mind is that subjects selected as controls must represent subjects that may turn into cases in the study (17, 26, 27). The number of those exposed to one or more of the risk factors studied in unhospitalized cases may be different from the number of those exposed to the same risk factors among the individuals comprising the population base that the cases come from. As a result, inclusion and exclusion criteria are generally used for controls that will prevent the under- or overrepresentation of conditions associated with the risk factors being studied and which might distort the representative nature of the controls. A list of inclusion and exclusion diagnostic conditions may also be established.

It is important to consider that the probability of an admitted subject being selected as a control is not connected to exposure, i.e, it does not depend on a positive or negative association with the factors being studied.

The advantages and disadvantages of using healthy or sick subjects as controls must also be considered. Sick subjects may have a better recollection of past events related to their disease. The researcher has a number of alternatives when it comes to selecting controls. One possibility is to conduct an unpaired study (27, 31) by obtaining a random sample of controls from the base population of the study, regardless of the characteristics of the cases. Another option is to conduct a paired study by selecting controls in accordance with one or more characteristics shared among the cases (26), such as age group, gender, etc. Pairing may be one-to-one (each control is paired to one case) or on a group basis (known as frequency pairing).

Pairing has several advantages (32): it increases efficiency (there is more useful information for analysis); it is useful in controlling known confounding factors; and it also protects against unknown or unmeasured confounding factors. When, for example, the distribution of cases by age is very diverse, pairing controls by age can ensure that the number of controls per case will be similar in all age groups, which will be useful for evaluating an effect in the different groups. When pairing is done using variables related to time (e.g., year of TB relapse, year of death, etc.), it makes it easier to compare the cases and controls in relation to exposures that vary in time.

One drawback is that pairing sometimes makes it more difficult to identify and obtain controls that meet the requirements (it takes longer or is more expensive). In addition, pairing also reduces the analysis possibilities as it is impossible to analyze the risk effect of a pairing variable because it is, by definition, similar in both the cases and the controls. It is, however, possible to measure the modification of the effect of exposure produced by the pairing variable. The variables most commonly recommended or to which pairing is applied are age, gender and race (21).

In multidrug-resistant TB, the controls may be TB patients in whom M. tuberculosis susceptible to antituberculous drugs and/or strains resistant to a single antituberculous agent have been isolated and identified. In our experience, pairing is more complicated in multicenter studies due to the difficulties of obtaining controls with the same age and gender characteristics as the cases.

Size of study, accuracy and power

In the design phase of a case-control study, the size of the study must be determined, i.e., the number of cases and the number of controls to be included. The study must be of sufficient size to prevent two types of errors: a or type I error and ß or type II error. The a error expresses the probability of concluding that there is a link to exposure when in reality there is not. ß error is defined as the probability of not detecting a real effect (the results of the study find there is no link when in fact there is).

The power of a study (in relation to size) is its capacity to reveal a link when this link really exists. The power is equal to the unit minus ß error: the higher the ß error, the lower the power. Accuracy is related to the reduction of random errors or errors caused by chance. Increasing the size of the study reduces the probability of there being an error due to chance. The larger the study, the greater the accuracy of the estimates and the lower the probability of finding false results due to chance.

However, increasing the size of a study does not increase its validity and may in fact reduce its efficiency. Validity is determined by the absence of systematic errors (in selection, information and confounding). Accuracy is linked to the exactness of the estimate of the effect, but not to validity. It is possible to conduct a very large study with limited validity, while a small study may be highly valid.

The elements that define the size of a study are the level of preestablished statistical significance (a error or type I error); the desired power; the expected number of exposed controls (the prevalence of exposure in the absence of disease); the magnitude of the expected effect (of the relative risk); and the ratio between the number of cases and the number of controls (33).

There are formulae for calculating the size of the study and there are also tables available (13) that make it possible to estimate size according to these parameters. It is also possible to work out the degree of power of a study given a number of cases and controls, an effect magnitude and a determined level of exposure among the controls. In general terms, the greater the magnitude of the effect and the higher the proportion of exposed controls, the smaller the study size required.

One of the components that defines the size of the study is the ratio of cases to controls, or in other words, the number of controls per case, which determines the total number of individuals involved in the study. This is of practical importance because when the number of available cases is relatively low, more controls per case may be included, thus maintaining the power of the study at the same level.

There are mathematical formulae that demonstrate that more than four controls per case gives only a minimal gain in power, meaning that it is advisable to include two, three and even four controls per case, but no more. Logically, when the number of available cases and controls for a study is high, and the cost and the difficulties in selecting, locating and interviewing them are similar, it is more reasonable to conduct the study with one control for each case.

The studies reviewed vary in sample size. They all use a small number of cases, but the number of controls can be similar to the number of cases, the total number of TB cases reported (18) or the total number of cases identified for which susceptibility to antimicrobials was tested (28).

Information gathering

The basic information in a case-control study is the past exposure of both the cases and the controls. The tools and markers used to define a category or level of exposure may vary in sensitivity and specificity.

The new fields of molecular biology and the various laboratory techniques to measure exposure are of great importance in the future of epidemiology (17, 18, 29). The biological indicators of exposure open up new horizons that may have a significant impact on the study of numerous risk factors, although they still raise many problems in the interpretation, validity and receptivity of the results, as well as in the standardization of laboratory techniques.

Classic tools used as sources of information include records (e.g., clinical histories, company records, insurance company records, etc.) and special questionnaires designed for the study. Questionnaires have been and remain the most common and useful means of gathering information.

Questionnaires for finding out about risk factors in resistance to antituberculous drugs may include open or closed questions. Closed questions may be more uniform and precise and may be easier to code and record. Questionnaires should be completed during an interview in person or over the phone, or may be completed by individuals and sent in by post. Self-administered questionnaires are easy to use with a large number of people, they reduce costs because there is no need for an interview, and they also reduce the amount of time required to gather the information. They also eliminate bias that the interviewer may introduce, but they can generate a very high proportion of "no response" answers and it is not possible to be certain about who really answered the questions.

The information to be gathered, the system used to collect it, the interview method and the structure of the questionnaire should be defined in the study design.

One of the classic limitations of case-control studies is that by studying the existence of exposure in the past, it may be difficult to obtain accurate, detailed information. Depending on the information to be obtained, it is possible that the subject may not remember distant events in detail. In order to avoid this, information from the recent past is gathered with the assumption that it is representative of the distant past.

The most important thing to bear in mind is that if there is an error in the information on exposure, both the cases and the controls are affected in a similar manner. This is the so-called nondifferential error in classifying exposure (34), which is always an underestimate of the effect of exposure. In contrast, if no effect is observed, this should always lead us to think that the negative result may be the consequence of the nondifferential error in classifying exposure.

The information gathered will depend on the aims of the study and on the risk factors being researched. The information should not only cover the risk factors being studied, but also other variables which may modify the effect or which may be potential confounding factors to be controlled in the analysis.

It is advisable to conduct a prior study on the validity of the tools or to use tools that have already been validated. In addition, conducting a pilot study on a small sample of subjects with characteristics similar to those of the overall study will make it possible to check the feasibility and viability of the information gathering techniques. A pilot study is essential when questionnaires and interviewers are used.

To avoid the "halo" effect in case interviews, the same interviewers must be used for the controls so that any systematic error introduced by an interviewer affects both the cases and the controls in a similar way. In addition, interviews with cases must be held at the same time as interviews with controls in order to eliminate any influence due to changes in the season or weather. It is also better to ensure that, in as much as possible, interviewers are unaware of the hypothesis of the study and of whether they are interviewing cases or controls so that they have no influence on the gathering of the information.

DATA ANALYSIS

Before starting the analysis phase of the study, it is necessary to check the quality of the information gathered through the questionnaires, and then codify it. In addition, a study of all the variables should initially be conducted, the minimum, maximum and aberrant values should be checked and the data rectified before beginning the analysis.

Measuring the effect

There are two possible measures of the effect of exposure in a case-control study: the relative measure and the absolute measure.

The relative measure of the effect is indicated by the RR, which measures the strength of the association, i.e., it measures how many times the disease (or effect being studied) is more frequent among the exposed than the unexposed. The RR is the rate ratio or relative rate as it expresses the ratio between the rate of incidence among the exposed in comparison with that among the unexposed.

Table1. Measuring effect in case control studies. 2 x 2 table for calculing the odds ratio (OR).