INT J TUBERC LUNG DIS 18(5):515–519 Q 2014 The Union http://dx.doi.org/10.5588/ijtld.13.0840
Latent tuberculous infection prevalence among paediatric contacts of drug-resistant and drug-susceptible cases R. Laniado-Labor´ın,*†‡ R. Cazares-Adame,*‡ M-L Volker-Soberanes,*‡ C. del Portillo-Mustieles,*‡ C. Villa-Rosas,*‡ L. Oceguera-Palao,*‡ J. Magallanes-Mijares* *Cl´ınica de Tuberculosis, Hospital General Tijuana, Tijuana, †Sistema Nacional de Investigadores, Consejo Nacional ´ de Ciencia y Tecnolog´ıa, Mexico City, ‡Facultad de Medicina y Psicolog´ıa, Universidad Autonoma de Baja California, Tijuana, Mexico SUMMARY
Tuberculosis (TB) clinic in Tijuana, M´exico. Tuberculin conversion rates in contacts of drug-resistant cases have been reported to be lower than among contacts of drug-susceptible cases. O B J E C T I V E : To determine if the prevalence of latent tuberculous infection (LTBI) among paediatric contacts of drug-resistant cases is lower than that of contacts of drug-susceptible cases. D E S I G N : In a cross-sectional study, LTBI among paediatric contacts of culture-proven TB cases was evaluated using the tuberculin skin test and interferongamma release assay. Rates among contacts of drugsusceptible and drug-resistant cases were compared.
The TST was positive in 83.1% of the drugsusceptible group vs. 76.0% for the drug-resistant group (P ¼ 0.25). Using the QuantiFERONw Gold In-Tube (QFT-GIT) assay, drug-resistant cases had a higher rate of positivity than the drug-susceptible group, although this difference did not reach statistical significance (42.3% positivity among drug-susceptible cases vs. 57.7% among drug-resistant cases; P ¼ 0.48). C O N C L U S I O N S : The proportion of LTBI is similar among paediatric contacts, regardless of whether the index case is drug-resistant or -susceptible. K E Y W O R D S : mycobacteria; mutation; tuberculin; IGRA
EARLY OBSERVATIONS suggested that streptomycin (SM) resistant Mycobacterium tuberculosis strains grew more slowly than their wild-type parents; the idea that changes in mycobacterial ‘fitness’ occurred on acquisition of resistance was later supported by animal studies.1 Molecular epidemiology studies have more recently reported that some drug-resistant M. tuberculosis isolates are less likely to be associated with disease transmission and clustering of cases than drug-susceptible isolates.2 Tuberculin conversion rates in contacts of drugresistant cases have also been reported to be lower than among contacts of drug-susceptible cases.3 However, the increasing number of human isolates of isoniazid (INH) resistant tubercle bacilli strongly suggests that some of these resistant organisms may retain much of their infectivity and virulence,4 and outbreaks of drug-resistant tuberculosis (TB) have raised concerns about the potential of drug-resistant strains of M. tuberculosis to spread widely and undermine global TB control efforts.2 Young children are at the highest risk of progressing to disease after infection; nonetheless, only a small number of studies
have investigated the rate of infection among children exposed to multidrug-resistant TB (MDR-TB).5 Our objective was to determine whether the prevalence of latent tuberculous infection (LTBI) among paediatric contacts of drug-resistant cases was lower than that of drug-susceptible cases.
SETTING:
BACKGROUND:
R E S U LT S :
SUBJECTS AND METHODS The state of Baja California has the highest rate of TB in Mexico (42.4 per 100 000 population) and the second highest number of MDR-TB cases in the country.6 In 2011, the state TB programme created a clinic for the referral of suspected or proven cases of drug-resistant TB. The TB Clinic is located in Tijuana, an urban centre (population reported at 2 million), with a high proportion of transient migrants due to its proximity to the United States. The clinic receives referrals from 32 health centres in the city, plus referrals from other cities in the state. Culture and drug susceptibility testing (DST) for first-line drugs are performed at the clinic (BACTECe MGITe 960 Mycobacterial Detection System, BD, Sparks, MD, USA); species identification is carried
Correspondence to: Rafael Laniado-Labor´ın, Cl´ınica de Tuberculosis, Hospital General Tijuana, Tijuana, Mexico. Tel/Fax: (þ52) 664 686 5626. e-mail:
[email protected] Article submitted 20 November 2013. Final version accepted 2 January 2014.
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out on solid media through conventional biochemical testing (niacin test). If required, samples are referred for second-line DST to the supranational TB laboratory in Mexico City. From August 2011 to June 2013, family contacts of culture-proven cases aged 616 years were tested for LTBI at the time of diagnosis of the index case using the QuantiFERONw Gold In-Tube assay (QFT-GIT) (QIAGEN Inc, Valencia, CA, USA) and the tuberculin skin test (TST) (5 tuberculin units purified protein derivative [PPD]; Tubersolw, Sanofi Pasteur Lt, Toronto, ON, Canada). Subjects with a history of TB, a previous diagnosis of LTBI or the administration of TST in the past year were excluded. Briefly, clinical information was obtained from every subject, including demographics, bacille Calmette-Gu´erin (BCG) vaccination record, culture results from the index case, exposure risk and physical examination (including verification of BCG scar). Blood was then drawn for QFT-GIT, followed immediately by a TST. TST was performed using the Mantoux method. An intradermal injection of 0.1 ml PPD was administered to the volar surface of the forearm. The transverse diameter of induration was recorded in mm 48 h after administration. An induration of 75 mm was considered positive, as every subject was a close contact of a culture-proven case.7 Each participant had 73 ml of blood drawn for QFTGIT, which was performed according to the manufacturer’s instructions. As recommended by the US Centers for Disease Control and Prevention (CDC),8 the QFT-GIT result was considered positive if the interferon-gamma response to TB antigens minus the negative control was 70.35 international units (IU)/ml and also .25% of the negative control, negative if these criteria were not met and indeterminate if either the negative control had a result of .8 IU/ml or the positive control had a result of ,0.5 IU/ml. Every contact with a positive test (either one), was referred to the paediatric TB clinic for a thorough clinical evaluation, including chest radiographs. The protocol study was approved by the Ethics Committee of the Tijuana General Hospital. Written informed consent was obtained from either parent of the children included in the study. Statistical analysis Data were analysed using SPSS version 19.0 (IBM, Chicago, IL, USA). Data were verified and validated before, during and after data entry using a data entry standard operating procedure. Categorical data were compared using the v2 test (or Fisher’s exact test when expected cell sizes were smaller than five). The Wilcoxon rank sum test was performed to determine whether the distribution of continuous variables differed between the two groups. Concordance
between the results of the TST and QFT-GIT tests was assessed using j coefficients for contacts with a drug-susceptible or a drug-resistant index case. Good agreement was defined as a j value between 0.4 and 0.6. Univariate analysis was performed on demographic, socio-economic and clinical characteristics, examining their association with drug-susceptible or -resistant TB in the index case. Logistic regression was used to estimate odds ratios (ORs) of positive responses for each of the variables measured. Variables included were age, sex, history of BCG vaccination, intensity of exposure, exposure time of the contacts to a source case, exposure to a drug-susceptible case and exposure to a drug-resistant case. All P values reported are based on twotailed comparisons, with statistical significance set at P , 0.05.
RESULTS Of the 173 subjects included, 77 were contacts of 37 drug-susceptible cases and 96 were contacts of 33 drug-resistant cases. The entire drug-resistant index cohort was smear-positive and had one or more lung cavities (only one patient was primary drug-resistant); only one index case among the drug-susceptible cohort was smear-negative and 34 had at least one lung cavity. Distribution of the contacts by sex was similar for both groups (51.9% males in the drug-susceptible group vs. 50.0% for the drugresistant group, P ¼ 0.87). There were no significant differences between the groups regarding medical history, history of BCG vaccination, exposure to the index case (h/day), number of rooms in the house, cohabitants and presence of signs or symptoms; all the subjects were asymptomatic at the time of the evaluation and none had active TB after being evaluated at the paediatric TB clinic. Contacts were treated for LTBI with INH or rifampicin (RMP) based on the culture results of the index case. Contacts of MDR-TB cases did not receive treatment for LTBI. Demographic and clinical variables are shown in Table 1. In the group of contacts exposed to a drug-resistant index case, the strain was monoresistant in 41 (42.7%), polyresistant in 14 (14.6%) and multidrug-resistant in 41 (42.7%). There were no significant differences in the proportion of positive reactors to TST or QFT-GIT. TST was positive in 83.1% of the drug-susceptible group vs. 76.0% of the drugresistant group (P ¼ 0.25). Using QFT-GIT, drugresistant cases had a higher rate of positive reactors than the drug-susceptible group, although this difference did not reach statistical significance (42.3% of the drug-susceptible had a positive test vs. 57.7% of the drug-resistant cases, P ¼ 0.48). Only one (0.57%) case was reported as indeterminate by the QFT-GIT test. Of the 101 QFT-GIT-negative subjects, 67
LTBI prevalence
Table 1
517
Demographic and clinical variables of drug-susceptible and drug-resistant groups
Variable
Drug-susceptible Mean 6 SD
Drug-resistant Mean 6 SD
Age, years Mean Median Range
7.79 6 4.28 8 1–16
7.36 6 4.46 7 1–16
Weight, kg
33.16 6 21.7
28.6 616.7
0.13
Height, cm Body mass index, kg/m2
125.2 6 27.3 18.9 6 4.9
121.2 6 28.4 18.0 6 4.4
0.34 0.20
BCG vaccination, %
94.8%
95.8%
0.75
Exposure to index case, h/day
12.6 6 6.1
13.9 6 7.6
0.24
Rooms in the house, n
2.56 6 1.1
2.57 6 1.0
0.93
Cohabitants, n
6.22 6 2.4
6.67 6 3.2
0.32
P 0.64
´ BCG ¼ bacille Calmette-Guerin.
(67.7%) were TST-positive, while only 2 of the 36 TST-negative subjects were QFT-GIT-positive (5.6%, P , 0.0001, j 0.27). In bivariate correlation analysis, only the TST induration diameter and the absolute value for QFTGIT were significantly correlated (r ¼ 0.47, P , 0.0001); however, the correlation was weak, as shown in the scatter graph (Figure), where 33 TSTpositive subjects had a QFT-GIT value of zero. Logistic regression analysis (dependent variable QFT-GIT results: negative 0, positive 1) for those bivariate correlations with P 6 0.10 is shown in Table 2. The only variables predictive of a positive QFT-GIT were older age and TST positivity. Logistic regression analysis with TST as a dependent variable had similar results, with a positive QFT-GIT test as the only predictor of a positive TST (results not shown). Stratification of the QFT-GIT and PPD results by extent of drug resistance is shown in Table 3. The analysis of variation test for differences between groups was significant for both tests (QFTGIT P ¼ 0.013 and PPD P ¼ 0.006).
DISCUSSION By the 1950s, it was already known that resistant M. tuberculosis isolates exhibited significantly less virulence than drug-susceptible strains.1,2 It was later found that mutations in the rpsL gene, which result in a high level of resistance to SM, can fall into one of two categories: restrictive and non-restrictive. Restrictive mutations are associated with an attenuation of virulence, whereas non-restrictive mutations are not.1 In the case of INH, experimental studies have shown that mutations or deletions within the katG gene result in reduced pathogenicity of INH-resistant M. tuberculosis strains.2 However, it is unclear how much of this reduction in guinea pig virulence applies to other experimental animals or to humans. The increasing number of human isolates of INH-resistant tubercle bacilli strongly suggests that some of these resistant organisms retain much of their infectivity and virulence.3 A recent study from China has shown that INH-resistant M. tuberculosis, particularly with the katG 315 Thr mutation, is likely to be clustered in a community, develops extra resistance to RMP and becomes MDR-TB in comparison to wild-type INH-resistant isolates, Table 2 Logistic regression analysis for predictive variables of a positive QFT-GIT (dependent variable QFT-GIT results: negative 0, positive 1) Variable
Figure Scatter graph showing correlation of QFT-GIT (absolute value) and TST induration in mm (r ¼ 0.47, P , 0.0001). QFT-GIT ¼ QuantiFERONW Gold In-Tube; TST ¼ tuberculin skin test.
Age, years Sex BGG vaccination Body mass index Exposure to source Hours/day exposure Cohabitants, n Rooms, n Tuberculin skin test Drug resistance
Significance 0.02 0.41 0.94 0.22 0.71 0.31 0.21 0.53 0.00 0.31
OR (95%CI) 1.12 0.74 0.94 0.94 0.91 1.03 0.91 1.12 19.3 1.43
(1.01–1.24) (0.37–1.50) (0.20–4.49) (0.85–1.03) (0.57–1.45) (0.96–1.10) (0.79–1.05) (0.77–1.61) (4.23–87.2) (0.71–2.90)
QFT-GIT ¼ QuantiFERONW Gold In-Tube; OR ¼ odds ratio; CI ¼ confidence ´ interval; BCG ¼ bacille Calmette-Guerin.
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Table 3
Stratification by extent of drug resistance
Drug resistance H Z S HþE HþZ HþR HþS HþZþS* HþRþS† HþRþZ HþRþZþS HþRþZþE HþRþZþEþS
QFT-GIT absolute value mean 6 SD 3.5 2.35 5.20 5.0 8.4 2.24 9.51
6 2.9 6 1.4 6 4.4 6 4.1 6 1.34 6 1.44 6 7.6 0.0 0.0 0.15 6 0.38 3.0 6 2.23 0.93 6 0.12 5.04 6 4.7
PPD induration (mm) mean 6 SD 13.6 14.2 20.3 15.0 22.5 11.0 23.1 8.5
6 10.6 610.6 6 11.0 6 14.0 6 3.5 6 7.1 6 15.4 6 0.7 30 11.2 6 10.6 9.0 6 8.8 14.1 6 11.1 24.7 6 9.0
* Two contacts in this group. † One contact in this group. QFT-GIT ¼ QuantiFERONW Gold In-Tube; PPD ¼ purified protein derivative; H ¼ isoniazid; Z ¼ pyrazinamide; S ¼ streptomycin; E ¼ ethambutol; R ¼ rifampicin.
evaluated through the more specific QFT-GIF in this population that was universally BCG-immunised, with rates of almost 60% among those exposed. One limitation and a potential source of bias in our study is that it is highly likely that contacts of drugresistant cases might have been exposed to an infectious source for a longer period of time (given that drug-resistant cases are usually longstanding) vs. drug-susceptible cases, which are usually new cases, and therefore exposure to an infectious source would be briefer. One notable finding is that there have been no active TB cases among the contacts after follow-up in the paediatric TB clinic from 6 months to 2 years, despite the fact that an overall prevalence of 2–4% is expected in this population.12
CONCLUSIONS suggesting strong transmissibility, full virulence and survival advantage under drug pressure in the host. This could be explained by the maintenance of the 30~40% catalase-peroxidase activity in the INHresistant strain with the katG 315 Thr mutation compared to other mutations related to drug resistance. Experimental studies using the model of guinea pigs infected with M. tuberculosis in which the katG gene was inactivated showed that the virulence of these strains was significantly reduced compared with that of the parent strain, and that it was restored when a functional katG gene was reintegrated into the genome.1 This emphasises the fact that the attenuation of virulence, if it occurs in INH-resistant organisms, only develops when the catalase is inactivated.9 Adaptation experiments with rpoB mutant strains of RMP-resistant M. tuberculosis show that, after passage for 88 generations, mutants that were initially less fit than the susceptible parent improved their fitness value to match and even exceed the parent. The assumption that resistant organisms are less fit than wild-type strains may not be correct, as the initial fitness deficits may be attenuated by adaptation by multiple passage.1 Although some drug-resistant M. tuberculosis strains appear to be less transmissible than susceptible isolates, it must be emphasised that MDR-TB strains of M. tuberculosis have caused several outbreaks. The high transmission rate of some MDR-TB strains has suggested that their fitness and virulence are unaffected, making these strains especially adapted to their hosts.10,11 Our results show that regardless of the test used to diagnose LTBI, paediatric contacts exposed to drugresistant cases had the same LTBI prevalence as contacts exposed to drug-susceptible cases. The rate of infection in both groups is very high, even when
The main finding in our study is that overall prevalence of LTBI in paediatric contacts in our region is high, and not significantly different among contacts of drug-susceptible and those of drugresistant patients. Thorough investigation of paediatric contacts should be carried out, regardless of whether the index case is drug-susceptible or -resistant. Conflict of interest: none declared.
References 1 Gillespie S H. Evolution of drug resistance in Mycobacterium tuberculosis: clinical and molecular perspective. Antimicrob Agents Chemoter 2002; 46: 267–274. 2 Daley C. Transmission of multidrug-resistant tuberculosis. Limited by man or nature? Am J Respir Crit Care Med 2000; 165: 742–743. 3 Nitta A T, Knowles L S, Kim J, et al. Limited transmission of multidrug-resistant tuberculosis despite a high proportion of infectious cases in Los Angeles County, California. Am J Respir Crit Care Med 2002; 165: 812–817. 4 Zhongming L, Kelley C, Collins F, Rouse D, Morris S. Expression of katG in Mycobacterium tuberculosis is associated with its growth and persistence in mice and guinea pigs. J Infect Dis 1998; 177: 1030–1035. 5 Seddon J A, Godfrey-Faussett P, Hesseling A C, Gie R P, Beyers N, Schaaf H S. Management of children exposed to multidrugresistant Mycobacterium tuberculosis. Lancet Infect Dis 2012; 12: 469–479. 6 National Center of Epidemiological Surveillance and Disease Control (CENAVECE). Sistema nacional de vigilancia epidemiologica. MexicoDP, Mexico : CENAVECE, 2013. /http:// ´ www.epidemiologia.salud.gob.mx/dgae/sinave/intd_sinave. html Accessed March 2014. 7 Targeted tuberculin testing and treatment of latent tuberculosis infection. This official statement of the American Thoracic Society was adopted by the ATS Board of Directors, July 1999. This is a Joint Statement of the American Thoracic Society (ATS) and the Centers for Disease Control and Prevention (CDC). This statement was endorsed by the Council of the Infectious Diseases Society of America. (IDSA), September
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1999, and the sections of this statement. Am J Respir Crit Care Med 2000; 161 (4 Pt 2): S221–S247. 8 Mazurek G H, Jereb J, Vernon A, LoBue P, Goldberg S, Castro K. Updated guidelines for using interferon gamma release assays to detect Mycobacterium tuberculosis infection - United States, 2010. MMWR Recomm Rep 2010; 13: 1–25. 9 Hu Y, Hoffner S, Jiang W, Wang W, Xu B. Extensive transmission of isoniazid resistant M. tuberculosis and its association with increased multidrug-resistant TB in two rural counties of eastern China: a molecular epidemiological study. BMC Infect Dis 2010; 10: 43.c 10 Soto C Y, Men´endez M C, P´erez E et al. IS6110 mediates
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increased transcription of the phoP virulence gene in a multidrug-resistant clinical isolate responsible for tuberculosis outbreaks. J Clin Microbiol 2004; 42: 212–219. 11 van Soolingen D, de Haas P E W, van Doorn H R, Kuijper E, Rinder H, Borgdorff M W. Mutations at amino acid position 315 of the katG gene are associated with high-level resistance to isoniazid, other drug resistance, and successful transmission of Mycobacterium tuberculosis in The Netherlands. J Infect Dis 2000; 182: 1788–1790. 12 Fox G J, Barry S E, Britton W J, Marks G B. Contact investigation for tuberculosis: a systematic review and metaanalysis. Eur Respir J 2013; 41: 140–156.
LTBI prevalence
i
RESUME
Centre anti-tuberculeux a` Tijuana au Mexique. C A D R E : Les taux de conversion du test a` la tuberculine (TST) ont souvent e´ t´e moins e´ lev´es chez les contacts de patients atteints de formes r´esistantes aux m´edicaments que chez ceux atteints de formes sensibles. O B J E C T I F : D´eterminer si la pre´ valence d’infection tuberculeuse latente (LTBI) chez les contacts p´ediatriques de patients r´esistants aux m´edicaments e´ tait plus faible que chez les contacts de patients sensibles aux m´edicaments. S C H E´ M A : Dans une e´ tude transversale, la LTBI chez des enfants au contact de patients pre´ sentant une tuberculose confirm´ee par culture a e´ t´e e´ valu´ee grace ˆ CONTEXTE :
au TST et au test de lib´eration de l’interf´eron-gamma. On a ensuite compar´e les taux de LTBI parmi les contacts de patients r´esistants et de patients sensibles. R E´ S U LT A T S : Le TST a e´ t´e positif chez 83,1% des enfants du groupe sensible contre 76% du groupe re´ sistant (P ¼ 0,25). En ce qui concerne le QuantiFERONw Gold In-Tube (QFT-GIT), les cas r´esistants avaient un taux plus e´ lev´e de positivit´e que le groupe sensible, bien que la diff´erence ne fut ˆ pas significative (42,3% des cas sensibles contre 57,7% des cas r´esistants, P ¼ 0,48). C O N C L U S I O N : La proportion de LTBI est comparable parmi les contacts p´ediatriques, quel que soit le statut (r´esistant ou sensible) du cas index. RESUMEN
M A R C O D E R E F E R E N C I A : Se ha reportado en la literatura que la tasa de infeccion ´ tuberculosa latente (LTBI) en contactos de casos de tuberculosis (TB) resistente a fa´rmacos es menor que la tasa de conversion ´ en contactos de casos con TB pansensible. O B J E T I V O: Determinar si esto es cierto en contactos pedia´tricos. ˜ D I S E NO Y M E´ T O D O S: Estudio de corte transversal llevado a cabo en la Cl´ınica de Tuberculosis del Hospital General Tijuana, en Tijuana, M´exico. Se investigo´ la presencia de LTBI en contactos pedia´tricos de casos de TB demostrada por cultivo mediante la prueba cuta´nea con tuberculina (TST) y un ensayo de
liberaci on ´ de interferon ´ gamma (QFT-GIT). Se compararon las tasas de conversion ´ entre los contactos de casos con cepas susceptible y casos con cepas resistentes. R E S U LT A D O S: La TST fue positive en 83,1% de los contactos susceptibles vs. 76,0% de los resistentes (P ¼ 0,25). Para el QFT-GIT los contactos resistentes tuvieron una tasa mayor que sin embargo no alcanzo´ significancia estad´ıstica (resistentes 57,7% vs. susceptibles 42,3%; P ¼ 0,48). C O N C L U S I O N : La tasa de LTBI en los contactos pedia´tricos es similar sin importar si el caso ´ındice presenta una cepa susceptible o resistante.
