Br Med Bull 1998 Eltringham 569 78

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Multiple drug resistant tuberculosis:

aetiology, diagnosis and outcome

I J Eltr ingham and F Drobniewski

Mycobacterial Reference Unit, PHL Dulwich, Dulwich Hospital, London, UK

Tuberculosis is an increasing problem worldwide both in terms of disease

burden and resistance to conventional antibiotic therapy. Studies of outbreaks

involving resistant strains have h ighligh ted th e need for b oth improved

infection control and the rapid provision of accurate susceptibility data. Each

pa tien t should undergo a risk assessment for possible resistance and those in

whom risk factors exist should be investigated by means of rapid molecular

techniques or other phenotypic methods, so tha t appropriate management can

be instituted with minimal delay. The ultimate outcome will vary according to

whether the patient is immunosuppressed, the tim e taken t o make a diagnosis,

the severity o f disease as well as the degree of resistance. The prognosis can be

improved w hen adequate a ntib iotic therap y is started as soon as resistance is

suspected. Adjuncts to conventional treatment such as surgery and perhaps

immuno therapy may be considered when response to antimicrob ial

chemotherapy has been subop timal.

Corresponding to

Dr IJ Eltringham

Mycobacterial Reference

Unit PHL Dulwich

Dulwich Hospital East

Dulwich Grove London

S

8QF UK

Tuberculosis (TB) is the leading cause of death due to an infectious

agent. It affects one-third of the world's population and 95% of the

disease burden is borne by developing countries whose economic and

healthcare infrastructures are often ill equipped to meet the demands

placed upon them

1

. Superimposed on this is the growing burden of HIV

infection, currently estimated to be over 22 million people and the

potential for re-activation of latent tuberculous infection in these

patients. Currently, the World Health Organization (WHO) estimates

that 6 million people are co-infected worldwide

2

. Not only are HTV

infected patients more likely to acquire TB after exposure, but the

annual rate of progression to disease approximates to the total lifetime

risk in non-HTV infected individuals

3

. In the US and Europe, annual

notification rates have increased since the mid 1980s and, although a

proportion of cases have co-incided with the development of the AIDS

pandemic, most of the excess cases have been seen in HTV negative

individuals suggesting other factors - such as increased mobility across

international boundaries, a declining public health infrastructure, and

poverty - are also responsible. The resurgence of what was considered

British Medical Bu lletin

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Resurgent/emergent infect ious diseases

to be a disease in decline prompted the WHO to declare TB to be a

global emergency in 1993 and subsequently to outline a strategy for

worldwide control which includes improved case detection and the

supervised administration of effective short course chemotherapy

4

.

Clearly, in order to improve control, these efforts must be directed

primarily at smear positive cases globally in order to reduce the pool of

infectious cases. Smear negativity, however, does not preclude trans-

mission, particularly in immunocompromised patients. With increasing

burden of disease comes the risk of antibiotic resistance. In patients

receiving inad equate therapy for T B, either as a result of no ncompliance

or ineffective regimens, the opportunity for overgrowth by resistant

organisms occurs with the potential for transmission to others. This

article focuses on drug resistance, as other aspects of TB are covered by

the article in this issue by Glynn.

Resistance to first line anti-tuberculosis drugs

The growth characteristics of

M ycobacterium tuberculosis,

the causative

organism of tuberculosis, are unusual in that it divides slowly both

in

vitro

and

in vivo,

is inherently resistant to many conven tional an tibiotics

and adapts to a number of different ecological niches

in vivo

5

.

The anti-

biotics selected to treat TB should penetrate macrophages and caseous

material and be effective against dormant subpopulations of organisms

which could be responsible for re-activation at a later date

5

. Rifampicin,

isoniazid and pyrazinamide are bactericidal agents which have good

sterilizing activity and when used in com bination p revent the emergence

of resistant mutants. Ethambutol and streptomycin are less effective

than other first line agents, although the addition of either to a regimen

is recommended until resistance to isoniazid can be excluded

6

. In vitro

studies show that resistant mutants arise spontaneously at a rate of

10~

5

-10~

8

organisms depending upon the antibiotic concerned

7

. The

total organism load in a cavitating lung lesion is likely to be in the region

of 10

9

, so if a single agent is used to treat clinical infection overgrowth

by resistant organisms is likely. The probability of resistance occurring

simultaneously to three agents in a fully compliant individual is

extremely small (circa 10~

20

). Resistance becomes clinically significant

when selective pressure is applied to spontaneous mutants by a com-

bination of patient and medical non compliance, incorrectly prescribed

drug regimens, or inadequate uptake of drugs.

The risk of primary resistance varies according to a patient's geo-

graphic location, ethnic group, HTV status and history of contact with

an individual infected with drug resistant TB. In general terms, higher

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Mu ltiple drug resistant tuberculosis

rates of primary resistance reflect a breakdown of TB treatment

programmes, although in practice this is not always clear because in many

individuals it is difficult to be certain that no previous therapy has been

given. In this context, the term initial resistance is used to include both

primary and undisclosed acquired resistance. Initial drug resistance is less

than 2% in UK born Caucasians without a history of contact with

resistant TB. Initial resistance to isoniazid is about 5% in black Africans

and individuals from the Indian subcontinent

6

. The inclusion of a fourth

agent is advisable when treating patients from areas with high initial

resistance rates for isoniazid. Single resistance to rifampicin, pyrazinamide

(excluding Mycobactertum bovts) and ethambutol are uncommon, but

may compromise successful treatment. Streptomycin resistance is quite

common in some countries, but the agent is not used on a regular basis in

the UK.

Multiple drug resistant

M. tuberculosis

(MDRTB) is resistance to at

least rifampicin and isoniazid. Of rifampicin resistant isolates seen at the

PHLS Mycobacteria Reference Unit (MRU), 90% are also resistant to

isoniazid. Rates of M DRTB in the UK have been increasing slowly over

the past two decades from approximately 0.6% of primary isolates in

the decade up to 1 991 to 1-2% of current isolates

8

-

9

. There is also wide

geographical variation, with the highest rates recorded in the North

Thames region

2

.

MDRTB strains referred to the MRU show considerable variation in

susceptibility to agents other than rifampicin and isoniazid and

frequently are isolated from patients who have lived outside the UK. At

least two major nosocomial outbreaks have occurred to date in the UK

and all but one of the patients involved were infected with HTV. This

situation differs from that in the US, where numerous outbreaks have

occurred in hospitals and rates of infection m hospital workers, assessed

by skin-test conversions have been high

10

.

Diagnosis of drug resistantM tuberculosis infection

The concept of clinical resistance is straightforward in that treatment is

likely to fail when an isolate is resistant to the agents used, hi micro-

biological terms, defining resistance is far more complex

7

. In practice,in

vitro

methods of measuring drug resistance fall into three groups: (i) the

absolute concentration method; (li) the proportion method; and (hi) the

resistance ratio method.

The technique currently used in the UK, and one of those adopted by

the WHO, is the resistance ratio method. It has been used in large

clinical trials and has proved to be a good predictor of clinical outcome

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Table 1 Known mutat ions conferring resistance in Mycobactenum tuberculosis

An t ib io t i c

Rifampic in

Isomazid

Pyrazinamide

Streptomycin

Eth ionamide

Ciprof loxacin

Target

RNA synthesis

Mycolic acid

synthesis

Protein synthesis

Cell wal l

DNA synthesis

M u t a t i o n

rpoB

katG

inhAJmabA

ahpC

oxyR

pncA

rrs

rpsL

inhA

gyrA

gyrB

Gene funct ion

DNA dependen t-

RNA polymerase

Catalase/peroxidase

Fatty acid biosynthesis

Alky lhydroperoxidase C

Oxidative stress regulator

Pyrazinamidase

16S rR NA

Ribosomal prote in SI2

Cross resistance with inhA

DNA gyrase

Molecular assay

PCR-SSCP, he ter od up lex

l ine probe, sequencing

PCR-SSCP, se qu en cin g

PCR-SSCP, se que nci ng

Sequencing

Sequencing

Sequencing



Sequencing


PCR = polymerase chain re actio n, SSCP = single strand co nfo rm atio nal polym orphis m

Adapted from Drobniewski and Pozniak

13

where resistance is demonstrated to one or more agents

11

. However,

most culture based techniques are relatively slow, although semi-

automated and automated liquid culture systems offer significant time

savings in generating drug susceptibility data. These rapid methods are

needed when a patient has significant risk factors for disease caused by

drug resistant organisms.

Direct susceptibility testing of clinical samples

In recent years, advances have been made in the detection of mutations

conferring resistance to a number of antibiotics. Telenti et at

12

demon-

strated the presence of point mutations in an 81 base pair segment of the

rpoB

gene, which codes for RNA polymerase. This has enabled several

genotypic methods for rifampicin resistance to be developed including a

commercial kit employing PCR amplification and solid phase

hybridisation to detect mutations in the product (see Table I)

13

. The test

has been applied to clinical samples and a high degree of correlation was

found with conventional methods

14

. Routine molecular determination of

resistance for other agents is less straightforward. For example, the

majority of pyrazinamide resistant isolates have mutations present in the

pncK

gene which codes for pyrazinamidase, an enzyme that converts

pyrazinamide into the bactericidal metabolite pyrazinoic acid. Although

572

British Medical Bulletin 1998,54 No 3)


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pncA

mutations are a good predictor of high level resistance, they are

scattered throughout the gene so that detection is less straightforward

than for rifampicin

15

. Mutations can also be demonstrated in isolates

resistant to isoniazid

13

but these occur in several genes, as summarized in

Table 1. In a large study of ethambutol resistance, a substitution at codon

306 of

embB,

which is involved in the biosynthesis of arabinogalactan,

was found in only 62% of resistant isolates suggesting that, as with other

agents, resistance is multifactorial

16

.

The heterogeneity of mutations conferring resistance has hindered the

development of routine molecular diagnostic tests for agents other than

rifampicin and rapid phenotypic methods which are not dependent on an

understanding of specific resistance mechanisms are now being developed.

One such technique involves the transfection of mycobacteria by specific

phages engineered to express the lux (luciferase) gene

17

. Infection of

antibiotic-resistant strains with this phage, in the presence of antibiotic,

results in the production of light as the bacteria continue to grow;

conversely, antibiotic-susceptible strains stop growing or fail to express

lux

and, thus, do not produce light. More simply, the presence of living

organisms can be demonstrated by infecting isolates with the phage, in the

presence of antibiotic, and looking for phage production from resistant,

but not susceptible, isolates using a lawn of rapidly growing

mycobacteria as the indicator strain (Phab assay)

18

. This test has been

shown to discriminate between sensitive and resistant strains for both

rifampicin and isoniazid and this method is now being applied to smear

positive sputum samples

16

. An alternative approach has been to use

mRNA as a marker of viability. Unlike DNA, mRNA has a short

half

life and is present in multiple copies, so its demonstration should

provide a sensitive indicator of viable organisms. This could be applied

to both susceptibility testing and microbiological response to treatment

in serial sputum samples. However, before novel genotypic and

phenotypic methods can be applied outside clinical research

laboratories, efforts need to be directed not only to standardizing test

parameters but also to improving cost effectiveness. Although

amplification based methods for rapid detection of mycobacteria are

sensitive and specific, they are unlikely to supercede culture based

methods in the immediate future.

In addition, a careful process of selecting those patients and samples

appropriate to a molecular investigation is of paramount importance, as

these techniques are designed to confirm a diagnosis, not as a screening

test for populations in whom the likelihood of tuberculosis is rare. False

positive results would limit the usefulness of any data generated under

these conditions

19

. It is equally important that molecular diagnosis

should only be carried out by institutions with expertise and well

defined quality control procedures, as wide variations in sensitivity and

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Resurgent/emergent infectious diseases

specificity have been demonstrated between a number of laboratories

with apparent exper t ise in PCR diagnosis

2 0

.

Outcome of treatment of multiple drug resistant tuberculosis

In MDRTB patients, the exclusion of first line agents with proven

effectiveness in clinical trials means that regimens of more toxic and less

effective drugs are used. Therapy should be individualised and guided by

drug susceptibility data, using bactericidal agents whenever possible, and

taking due consideration of potential side effects and drug interactions.

The effectiveness of therapy should be judged by sputum smear and

culture conversion and, if sputum remains smear or culture positive

beyond 3-4 months in a patient whose clinical features are not resolving,

serious consideration should be given to repeating susceptibility tests and

making a therapeutic switch, substituting two, or preferably three, drugs

for agents not used previously. Anecdotal evidence suggests that relapse is

unacceptably high in MDRTB if less than 18-24 months of appropriate

therapy is used beyond culture conversion

11

. In HIV-positive patients, it

may be advisable to continue therapy for life, although bacteriological

and clinical cure can be demonstrated in some individuals, particularly

where effective agents are employed withm a month of diagnosis. When

choosing a regimen to treat a patient with MDRTB, it is sensible to

consider the following points

3

: (i) as many first line agents to which the

organ ism is sensitive should be used; (ii) the inclusion of paren teral agents

reduces problems with compliance and absorption; ( iii) administration of

oral agents shou ld be directly obs erved ; (iv) wh enev er po ssible

bactericidal agents should be used; (v) response and survival are more

likely if at least three agents to which the isolate is sensitive are used; and

(vi) single agents should never be added to a failing regimen.

In addition, combinations of agents whose side effect profiles are

similar should be avoided, monitoring of drug levels may be needed and,

in all instances, therapy should be monitored by physicians with

exper t ise in t rea t ing MDRTB cases.

Surgery is an option for some patients. In a large study, 57 patients with

MDRTB were treated surgically m addition to receiving a number of

chemotherapeutic regimens. Most were smear positive at the time of

surgery but, following pneumonectomy or lobectomy, over 80% of

patients remained smear negative at 36 months

11

. In another study,

however, artificial pneumoperitoneum was carried out in a series of end

stage MDRTB cases and no clear benefit could be demonstrated

2 1

. The

role of surgery is less clear in HIV-positive patients where disease is often

widespread. However, in some individuals with localized infection,

surgery may offer the only chance of surviving the disease.

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Multiple drug resistant tuberculosis

Studies of TB patients given immunotherapy with Mycobacterium

vacate

have yielded equivocal results. One poorly controlled study in

northern Nigeria showed improvement in both clinical and micro-

biological response in those givenM.vaccae 1-3 weeks after commence-

ment of quadruple therapy

22

. Follow-up of cases in M.

vaccae

treated

individuals in Romania failed to show any statistically significant

differences in terms of outcome compared to p lacebo

23

. Some benefit was

gained in patients with chronic, relapsing disease given immunotherapy,

where the chances of a successful outcome rose from 44% to 63% in

those given a single dose of M.vaccae at 1 month

24

. Recent preliminary

data from the South African study using

M.

vaccae suggest that results

have been disappointing, with no benefit shown over the application of

directly observed chemotherapy

25

.

Until more conclusive data of clinical efficacy are available from

ongoing double blind placebo controlled trials, it would be reasonable to

restrict immunotherapy to drug resistant cases where conventional

methods have failed.

Although published data on the use of immunomodulating cytokines

are limited in MDRTB pa tients, one small study showed that some benefit

may be afforded when appropriate chemotherapy is augmented w ith aero-

solised interferon gamma in patients with pulmonary disease

26

. However,

as with M. vaccae,more da ta from well controlled studies are needed

before cytokine therapy can be recommended outside clinical trials.

Outcome of treatment and prevention of transmission

Results from two large follow up studies in the US showed that a

favourable clinical outcome can be expected in the majority of HTV-

negative patients infected with MDRTB, although the prognosis was

worse when disease had been present for a number of years and thera-

peutic options were limited

3

. One crucial factor determining outcome is

the time taken to institute appropriate therapy. Risk factor assessment

must be carried out in all patients who are to receive treatment, and the

clinical history should identify any contact with a known resistant case,

previous TB therapy, and HIV status. An inadequate clinical response,

such as persistence of pyrexia beyond 2 weeks of appropriate first-line

therapy, is suggestive of treatment failure which may be due to M DR TB.

Outcome is less favourable in patients co-infected with HTV. Median

survival rates of 78-315 days have been reported in HIV-positive

individuals

3

. Survival is significantly reduced when patients have AIDS

and sputum conversion is rare in these individuals

11

.

Recent nosocomial outbreaks have resulted from failures in simple

infection control. In the UK, experience with MDRTB in nosocomial


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Resurgent/emergent infectious diseases

outbreaks has been limited to two incidents involving relatively few

patients. In both instances, problems were subsequently found in the

implementation of infection control measures. In contrast, by 1994 the

US had already witnessed over 100 MDRTB infections in patients, and

16 cases in healthcare workers, occurring as a result of nosocomial

transmission

10

. In one outbreak, the results of intervention in the form

of tightening of infection control practices were analysed. Of new cases,

80% were attributable to nosocomial spread before intervention, but

this fell to zero when guidelines issued by the US Centers for Disease

Control were followed

10

. The release of MDRTB infected patients from

isolation is frequently a controversial issue. Patients whose sputum is

repeatedly culture negative may be treated on an outpatient basis, but

therapy must continue to be directly observed and the patient's clinical

condition closely monitored on follow-up, as up to 20 % of patients may

relapse following sputum conversion if therapy is not prolonged

11

.

Conclusions

The global resurgence of TB has been accompanied by increases in the

rates of antibiotic resistance. The majority of resistance is due to the

selection of small populations of naturally occurring mutants during

exposure to inappropriate regimens.

In 1995, it was estimated that less than one-quarter of the world's

population was covered by control programmes that followed the WHO

strategy and accurate figures for the number of patients being treated for

TB ,who have a favourable outcome, are not available. Estimates range

from 37% to 78

27

.

Com prehensive data on the existence and ex tent of

drug resistance around the globe have been lacking

28

, but a recent

monograph from the WHO quoted drug resistance data from 35

national surveillance programmes and median rates for primary

resistance in isolates ranged from 0% to 10.8%

29

.

Patients with suspected or proven MDRTB should be managed by a

multidisciplinary team involving chest, infectious disease, and HIV

physicians, as well as clinical microbiologists with experience in both

treatment, diagnosis and infection control

3

. Outbreaks of MDRTB

infection should not occur if current guidelines are followed but, when

they are suspected, clusters should be confirmed by molecular

techniques, such as restriction fragment length polymorphism analysis

or spoligotyping

9

. This enables the team to monitor the course and

extent of an outbreak as well as assessing the outcome of any

intervention.

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Br Med Bull 1998 Eltringham 569 78

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