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Emerging Infectious Diseases
Volume 2, Number 2, April-June 1996

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Dispatches

Nosocomial Transmission of Multidrug-Resistant Mycobacterium tuberculosis
in Spain

Before 1990, outbreaks of multidrug-resistant tuberculosis (MDRTB) were
uncommon (1); since then, more than 10 outbreaks have been reported, all in
hospitals and prisons in the eastern United States (2-7). Persons
traditionally considered at risk for MDRTB (foreign-born TB patients and
those inadequately treated for TB) have not been associated with these
outbreaks. Instead, the presence of patients with active TB near
immunocompromised patients in HIV-dedicated wards has led to MDRTB-infected
HIV patients whose TB cases often go unrecognized. The patients receive
inadequate treatment in facilities without effective procedures for
isolating acid-fast bacilli; these circumstances favor nosocomial
transmission. Health officials in other geographic areas where HIV and TB
are major public health threats have been alerted to this emerging problem,
and surveillance systems have been designed (8).

Spain has the highest reported incidence rate of AIDS in Europe (143.4
cases per million in 1994) (9). Although in Spain TB is not notifiable at
the national level, reported rates in the autonomous community of Madrid
for 1994 were among the highest in Europe (33.5/100,000) (10). During a
45-month period starting in September 1991, a number of patients and one
health care worker became infected with MDRTB in a 120-bed, infectious
disease reference hospital in urban Madrid. In May 1995, the Field
Epidemiology Training Program of the Spanish Ministry of Health was invited
to assist the Madrid Department of Health and hospital officials in
investigating the outbreak. This report describes the findings of the
epidemiologic and molecular laboratory investigation and analyzes risk
factors associated with the outbreak. The study was designed in three
parts: 1) a description of the reported MDRTB cases, including a laboratory
investigation of isolates; 2) a case-control study comparing HIV-infected
patients who also had MDRTB to HIV-infected patients who did not have
MDRTB; and 3) a study of tuberculin conversion among hospital employees.

We reviewed the medical records and laboratory specimen testing results of
a series of patients with MDRTB in the HIV-dedicated ward of the hospital
for January 1991 through June 1995. Cases were defined as patients with
culture-confirmed TB and drug resistance to at least rifampin and isoniazid
and with no previous history of inadequate treatment. Demographic and
clinical variables were collected to characterize the cluster. Drug
susceptibility testing and DNA subtyping analysis were performed with
resistant strains available at the time of the study. Drug susceptibility
testing was performed by the method of proportions in Middlebrook 7H11
medium distributed in petri plate compartments (reference).

All 48 reported cases of isoniazid and rifampin resistance were among
HIV-infected patients hospitalized in the HIV-dedicated ward from September
15, 1991, to May 1995. One patient was an HIV-infected nurse who worked on
the ward from 1990 to 1994. The mean age of patients was 34.1; 81.3% were
male, and 66.6% were intravenous-drug users (Table 1). Of the 47 (97.9%)
who died, the mean interval from diagnosis to death was 77.6 days.


 Table 1. MDRTB patient characteristics
----------------------------------------------
                   Number
     Variable       (n =       %
                    48)
----------------------------------------------
 Sex

  Male               39       81.3

  Female              9       18.7

 Mean age            34.1     (6.8)a

 HIV status

  Infected           48      100.0

 HIV risk group

  Intravenous
  drug users         32       66.6

  Homosexual         10       20.8

  Others              6       12.6

 Outcome

  Death              47       95.9

  Discharge           1        4.1

 Mean survival
 after                      (107.8)a
 MDRTB diagnosis     77.6
 (in days)
----------------------------------------------
 a Standard deviation.

The epidemiologic curve suggests a propagated transmission pattern of MDRTB
among HIV ward patients, starting in 1991 and continuing until June 1995
(Figure 1). By the first 6 months of 1995, 65% of Mycobacterium
tuberculosis strains seen among HIV ward patients were multi
drug-resistant.

[Image not available in ASCII version]
Figure 1. Hospital outbreak, Madrid, Spain, 1995

At the beginning of the outbreak, no consistent antibiogram pattern was
observed among isolates. However, beginning in 1993, strains were
consistently resistant to isoniazid, streptomycin, ethambutol, and rifampin
(HSER); of the 26 patients with an MDRTB diagnosis since the last trimester
of 1993, 24 (92.3%) had HSER isolates. DNA subtyping analysis was performed
on 12 of these HSER isolates that were available. Eleven of the12 strains
had the same band patterns (Figure 2). Two additional isolates with a
different antibiogram pattern had different banding patterns.

[Image not available in ASCII version]
Figure 2. Evolution of MDRTB strain* over time, hospital outbreak, Madrid,
Spain, 1995.

Subtyping of M. tuberculosis strains was performed (11) by analyzing DNA
located between two copies of repetitive sequence IS6110. A 10-l sample of
the extracted DNA was amplified in a reaction mixture containing 0.5 pM of
each of the four primers (Ris1, Ris2, Pntb1, and Pntb2), 200 UM DNTPs, 50
mM Tris-HCL, 50 mM KCL (pH 8.8), 2.5 mM MgC12, 0.1% Triton x-100, and 0.5%
Taq polymerase. The samples were denatured by incubation at 95C for 10 min
and amplified by 30 cycles of denaturation at 94C for 1 min, primer
alignment at 56C for 2 min, and primer extension at 72C for 1 min. The
amplification products were analyzed by electrophoresis in 2% agarose gel
stained with ethidium bromide and observed with an ultraviolet
transilluminator.

HIV-infected patients hospitalized on the HIV-dedicated ward between
September 15, 1991, and December 31, 1994, who had TB diagnosed in 1994
with a known drug-susceptibility pattern were included in a case-control
study. Case patients isolates were resistant to isoniazid, rifampin,
streptomycin, and ethambutol; control patients isolates were sensitive to
the same antimicrobial drugs.

A time-person line diagram was prepared for each of the case patients and
control patients, including all hospitalization dates, ward and room
number, potentially infectious days, and possible days exposed to infective
patients. Prior admission was defined as all admissions to the
HIV-dedicated ward since the MDRTB patient was diagnosed (9/15/91) until
the end of the period (12/31/94). TB patients were classified as
potentially infective from the 2 week-period before sputum or culture
results were positive for M. tuberculosis until the time sputum results
were negative or the patient died. TB-negative patients were classified as
possibly exposed if they were hospitalized on the HIV-dedicated ward
during the time a potentially infective patient was also present, beginning
on September 15, 1991, until 2 weeks before TB diagnosis.

Thirty-five patients (18 with cases and 17 controls) met the established
study case/control definition. Case patients and control patients were not
significantly different with respect to age, sex, HIV risk factors,
interval between HIV and TB diagnoses or count of CD4 lymphocytes at the
time of TB diagnosis (Table 2).

          Table 2. Population characteristics: case control study
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                                         Control patients
                     Case patients (%)         (%)
      Variable            n = 18              n = 17         OR (95% CI)a
----------------------------------------------------------------------------
 Mean age (yr)          34.4              35.2                 p = 0.30b

 Male sex               13 (72.2)         14 (82.3)            1.8 (0.3-12.1)

 IVDU                   11 (61.1)         11 (64.7)            1.2 (0.2-6.7)

 Interval HIV to TB                                         p = 0.79b
 diagnosis in days    1403              1293

 CD4 lymphocyte                                             p = 0.30b
 count, median         112.9             199.5
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 a OR (95% CI) = odds ratio (95% confidence intervals); Fishers Exact Test.
 b p value for analysis of variance (ANOVA). IVDU = intravenous drug user.

Before the hospitalization during which TB was diagnosed, 76.4% of the
case-patients had been hospitalized on the HIV-dedicated ward of the
hospital versus 23.5% of control patients (OR = 7.8 [1.4,50.5]) (Table 3).
Of the five case patients with no prior hospitalization, three were family
members of previously hospitalized HIV-infected patients who had visited
the HIV ward frequently during the outbreak period.

Case patients and controls were compared with respect to possible exposure
to a potentially infective wardmate beginning on 9/15/91 until 2 weeks
prior to the diagnosis of TB. Case patients were more likely to have been
exposed to potentially infective wardmates (72.2% with a median of 26.4
days) than control patients (41.2% with a median of 7.6 days). When we
stratified possible ward exposure by days (0 days vs. 1 to 30 days vs. 30
days), we observed a dose-response effect, and the chi-square for linear
trend was statistically significant (Table 3). Case patients were more
likely to die during their initial hospitalization for MDRTB(94.4%) than
were control patients (29.4%) during their hospitalization for TB (OR =
40.8 [3.6,1842]) (Table 3).

    Table 3. Variables associated with MDRTB
----------------------------------------------------
                Case      Control
              patients   patients
                 (%)        (%)
  Variable     n = 18     n = 17      OR (95%
----------------------------------------------------
 Prior
 admission
 to HIV ward

  Yes        13 (76.4)    5 (29.4)    6.2
                                    (1.2-37.2)

  No          5 (23.6)   12 (70.6)

 Possible
 ward
 exposure
 days

  None        5 (27.8)   10 (58.8)    1.0

  1-30        6 (33.3)    5 (29.4)    2.4

                                      7.0, p =
  30          7 (38.9)    2 (11.8)    0.03b

 Outcome

  Death      17 (94.4)    5 (29.4)   40.8
                                     (3.6-1842)

  Discharge   1 (5.6)    12 (70.6)
----------------------------------------------------
 a OR (95% CI) = odds ratio (95% confidence
 intervals); Fishers Exact Test.
 b Chi-square for linear trend.

A TB screening clinic visit was offered to all hospital employees after the
outbreak was identified. Of the 591 active employees, 565 (95.6%)
participated. Of these, 288 (51%) had not participated in previous hospital
employee screening programs conducted in 1990 and 1994. The overall
prevalence of TB infection among participating employeeswas 450 (80%) of
565; only 115 (20%) of the current employees tested were
tuberculin-negative.

Employees currently working at the hospital, who had a documented negative
(< 6 mm) tuberculin test between January 1993 and June 1995 were eligible
for this skin test conversion study. Many of the employees had received BCG
vaccine. For those who had not received BCG vaccine, conversion was defined
as an induration of 10 mm or greater with a change of at least 6 mm of
induration since the last negative tuberculin test (12). For BCG vaccinees,
conversion was defined as a 15-mm induration change since the last negative
(< 6 mm) skin test.

Employees were defined as occupationally exposed if they worked in parts of
the hospital where exposure to patients or M. tuberculosis was likely (the
HIV-dedicated ward, HIV outpatient clinic, radiology unit, the
mycobacteriology laboratory, and the internal medicine ward). Employees
were asked to quantify the cumulative number of months spent in these
high-risk areas, regardless of their usual place of work, during the
30-month study period.

According to the Mantoux technique, 2 tuberculin units of purified protein
derivative (PPD) tuberculin RT-23 were administered in the anterior forearm
of the screened employees, and the results were read within 48 to 72 h. Of
the participants, 92 (16.3%) were eligible for the conversion study. The
incidence of conversion during the 30-month period was 24 of 92 (26%).
Employees who had occupational exposure to high-risk areas had higher
conversion rates than employees who did not have occupational exposure to
high-risk areas (RR = 5.0 [2.7,9.6]) (Table 4). The conversion had a
dose-response effect, that is, the more months the person is occupationally
exposed to high risk-areas, the higher the risk for conversion.

     Table 4. Employee tuberculin screening
            conversion study results
----------------------------------------------------
               Convertors  Nonconvertors    RR
                  (%)           (%)        (95%
   Variable      n = 24       n = 68       Cl)
----------------------------------------------------
 Occupational
 exposure to
 high-risk
 areas

                                           5.0
      Yes      14 (70.0)      6 (30.0)    (2.7,
                                           9.6)

       No      10 (13.9)     62 (86.1)

 Months
 exposed                                   ORb
 in high-risk  
 areas

       0        6 (10.7)     50 (89.3)     1.0

      1-6       4 (28.6)     10 (71.4)     3.3

                                          14.6,
      7-36     14 (63.6)      8 (36.4)      p <
                                           0.01
----------------------------------------------------
 a RR (95% CI), relative risk (95% confidence
 intervals).
 b Chi-square for linear trend.

This is the first nosocomially transmitted MDRTB outbreak reported in
Spain, which, with recent outbreaks in the United Kingdom (13) and Italy,
is among the first in Europe. Its characteristics are similar to the other
reported outbreaks on that it occurred in an HIV-dedicated ward among
non-foreign-born patients who had not been treated for TB; it had a high
mortality rate within 3 months of onset during which mycobacteria
laboratory surveillance recognized similar antibiogram resistant patterns;
and identification of MDRTB isolates was followed by DNA subtyping, which
confirmed that the same strain was responsible for the outbreak. Risk
factors identified include admission to the HIV-dedicated ward and more
possible exposure days to potentially infective wardmates; additionally,
skin test conversion rates among employees were directly related to working
in high risk areas of the hospital. The consistency of these findings with
those reported in similar outbreaks and the fact that the same isolate was
cultured over an extensive period among many different patients on a
hospital ward without proper room isolation techniques support the
conclusion that nosocomial transmission was the leading cause of the
outbreak.

CDCs recommended guidelines for hospital TB prevention and control (14)
were not fully implemented in the hospital. Acid-fast bacilli room
isolation techniques were not in place; moreover, no ventilation system was
available to provide negative pressure to prevent bacilli from passing from
the MDRTB patients rooms to the hallway or to provide the six air
interchanges per hour recommended for removing bacilli from room air.
Surgical masks used in the HIV-dedicated ward during the outbreak as
protective masks are not recommended for this purpose because of filtering
and facial sealing problems. Observational visits to the HIV-dedicated ward
revealed that MDRTB patients without masks were walking, talking, and
smoking in the halls and in the lounge next to the unprotected patients,
families, and staff.

Complete follow-up of employees is an essential component of a hospital
control program (14). Because 80% of the employees in this outbreak were
tuberculin-positive,chest radiographs became an important component for
disease screening; yearly surveillance of the tuberculin-negative employees
will help determine if the interventions in place to prevent future
outbreaks have worked in this setting.

Once the case-control study findings were analyzed, a TB control committee
was established. All MDRTB patients were placed in a separate area of the
hospital. Hospital staff were informed about the outbreak and alerted about
future possible cases and the patient management and treatment schemes. The
mycobacteria laboratory expanded the antibiogram service to include all
second-line antibiotics used by the hospital clinicians. Clinicians have
elaborated a treatment flow chart for MDRTB patients. Masks that fulfilled
the sealage and filtering criteria (15) were purchased, and mask use was
aggressively implemented. An employee health clinic was also instituted,
with a prophylaxis and chest X-ray follow-up program for the 410 infected
employees, along with a computerized follow-up surveillance system which
included all employees graded by occupational risk category. Plans were
made to screen tuberculin-negative employees every 3 months to identify
those who had recently seroconverted. Family, community members and
wardmates of all patients whose MDRTB had been diagnosed within the
previous 6 months were notified of their risk and were scheduled for
follow-up evaluations. The HIV-dedicated ward will be transformed into an
acid-fast bacilli isolation zone, with an exclusive ventilation system that
provides 11 individual rooms, each with 12 air interchanges per hour and
negative pressure relative to the hallway.

Health officials in Europe need to be updated about this emerging problem,
especially in areas of high HIV and TB prevalence. Basic universal TB
control and prevention measures (16,17) should be implemented by all
general community hospitals in Spain, especially those with HIV-dedicated
wards in areas where TB is prevalent. TB surveillance of health care
employees is necessary to identify emerging problems as well as to protect
employees, patients, and visitors.

John V. Rulln M.D.,* Dionisio Herrera, M.D.,* Rosa Cano, M.D.,* Victoria
Moreno, M.D., Pere Godoy, M.D.,* Enrique F. Peir, M.D.,* Juan Castell,
M.D.,* Consuelo Ibaez, M.D.,* Arturo Ortega, M.D., Leopoldo Snchez
Agudo, M.D., and Francisco Pozo, M.D.

Instituto de Salud Carlos III, Ministerio de Sanidad y Consumo: *Programa
de Epidemiologa Aplicada de Campo-Centro Nacional de Epidemiologa;
Centro Nacional de Investigacin Clnica y Medicina Preventiva;
Subdireccin General de Salud, Madrid, Spain

Drs. Herrera, Peir, Castell, and Godoy have received a scholarship from
the Fondo de Investigacin Sanitaria del Instituto de Salud Carlos III
during their 2-year epidemiologic training period in the Field Epidemiology
Training Program (Programa de Epidemiologa Aplicada de Campo).

Acknowledgments

We thank the staff of the Center for Clinical Investigation and Preventive
Medicine of the Instituto de Salud Carlos III for their collaboration in
this investigation; in particular, staff from the following departments:
Administration, Respiratory Diseases, Infectious Diseases, Nursing, and the
Mycobacteriology Laboratory. For the employee tuberculin study, we thank
Drs. Rafael Rey, and Dura; Leopoldo Sanchez Agudo; Margarita Muoz Arcaiz;
Consuelo Yubero Higuera; Concepcin Gonzalez de la Calle; Lucia Martin
Gadea; and Pilar Fuertes Rodrigues for their collaboration.We acknowledge
Dr. Jack Crawford and staff from the Division of Bacterial and Mycotic
Diseases, National Center for Infectious Diseases, CDC, for their
collaboration as reference laboratory for DNA subtyping analysis; Dr.
Daniel Fishbein for editing the manuscript; and Dr. Rafael ReyDurn for
providing us with the 1990 and 1994 data set of the hospital employees
tuberculin test screening. We thank Luis Dorado for his graphics
collaboration.

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