Utility of the QuantiFERON-TB Gold In-Tube assay for the diagnosis of tuberculosis in Moroccan children.

SETTING: The utility of interferon-gamma release assays (IGRAs), such as the QuantiFERON-TB Gold In-Tube (QFT-GIT) test, in diagnosing active tuberculosis (TB) in children is unclear and depends on the epidemiological setting. OBJECTIVE: To evaluate the performance of QFT-GIT for TB diagnosis in children living in Morocco, an intermediate TB incidence country with high bacille Calmette-Gurin vaccination coverage. DESIGN: We prospectively recruited 109 Moroccan children hospitalised for clinically suspected TB, all of whom were tested using QFT-GIT. RESULTS: For 81 of the 109 children, the final diagnosis was TB. The remaining 28 children did not have TB. QFT-GIT had a sensitivity of 66% (95%CI 5277) for the diagnosis of TB, and a specificity of 100% (95%CI 88100). The tuberculin skin test (TST) had lower sensitivity, at 46% (95%CI 3360), and its concordance with QFT-GIT was limited (69%). Combining QFT-GIT and TST results increased sensitivity to 83% (95%CI 6992). CONCLUSION: In epidemiological settings such as those found in Morocco, QFT-GIT is more sensitive than the TST for active TB diagnosis in children. Combining the TST and QFT-GIT would be useful for the diagnosis of active TB in children, in combination with clinical, radiological and laboratory data.

A genome-wide association study of pulmonary tuberculosis in Morocco.

Although epidemiological evidence suggests a human genetic basis of pulmonary tuberculosis (PTB) susceptibility, the identification of specific genes and alleles influencing PTB risk has proven to be difficult. Previous genome-wide association (GWA) studies have identified only three novel loci with modest effect sizes in sub-Saharan African and Russian populations. We performed a GWA study of 550,352 autosomal SNPs in a family-based discovery Moroccan sample (on the full population and on the subset with PTB diagnosis at <25 years), which identified 143 SNPs with p < 1 × 10(-4). The replication study in an independent case/control sample identified four SNPs displaying a p < 0.01 implicating the same risk allele. In the combined sample including 556 PTB subjects and 650 controls these four SNPs showed suggestive association (2 × 10(-6) < p < 4 × 10(-5)): rs358793 and rs17590261 were intergenic, while rs6786408 and rs916943 were located in introns of FOXP1 and AGMO, respectively. Both genes are involved in the function of macrophages, which are the site of latency and reactivation of Mycobacterium tuberculosis. The most significant finding (p = 2 × 10(-6)) was obtained for the AGMO SNP in an early (<25 years) age-at-onset subset, confirming the importance of considering age-at-onset to decipher the genetic basis of PTB. Although only suggestive, these findings highlight several avenues for future research in the human genetics of PTB.

Pott's disease in Moroccan children: clinical features and investigation of the interleukin-12/interferon-γ pathway.

SETTING: Tuberculosis spondylodiscitis (TS), or Pott's disease, an extra-pulmonary form of tuberculosis (TB), is rare and difficult to diagnose in children. Some cases of severe TB in children were recently explained by inborn errors of immunity affecting the interleukin-12/interferon-gamma (IL-12/IFN-γ) axis. OBJECTIVE: To analyse clinical data on Moroccan children with TS, and to perform immunological and genetic explorations of the IL-12/IFN-γ axis. DESIGN: We studied nine children with TS diagnosed between 2012 and 2014. We investigated the IL-12/IFN-γ circuit by both whole-blood assays and sequencing of the coding regions of 14 core genes of this pathway. RESULTS: A diagnosis of TS was based on a combination of clinical, biological, histological and radiological data. QuantiFERON(®)-TB Gold In-Tube results were positive in 75% of patients. Whole-blood assays showed normal IL-12 and IFN-γ production in all but one patient, who displayed impaired decreased response to IL-12. No candidate disease-causing mutations were detected in the exonic regions of the 14 genes. CONCLUSIONS: TS diagnosis in children remains challenging, and is based largely on imaging. Further investigations of TS in children are required to determine the role of genetic defects in pathways that may or may not be related to the IL-12/IFN-γ axis.

[Human genetics of tuberculosis]. / Génétique humaine de la tuberculose.

Tuberculosis (TB), caused by Mycobacterium tuberculosis, remains a major public health problem worldwide, resulting in 8.7 million new cases and 1.4 million deaths each year. One third of the world's population is exposed to M. tuberculosis and, after exposure, most, but not all, individuals become infected. Among infected subjects, only a minority (∼10%) will eventually develop clinical disease, which is typically either a primary, often extra-pulmonary, TB in children, or a reactivation, pulmonary TB in adults. Considerable genetic epidemiological evidence has accumulated to support a major role for human genetic factors in the development of TB. Numerous association studies with various candidate genes have been conducted in pulmonary TB, with very few consistent results. Recent genome-wide association studies revealed only a modest role for two inter-genic polymorphisms. However, a first major locus for pulmonary TB was mapped to chromosome 8q12-q13 in a Moroccan population after a genome-wide linkage screen. Using a similar strategy, two other major loci controlling TB infection were recently identified. While the precise identification of these major genes is ongoing, the other fascinating observation of these last years was the demonstration that TB can also reflect a Mendelian predisposition. Following the findings obtained in the syndrome of Mendelian susceptibility to mycobacterial diseases, several children with complete IL-12Rß1 deficiency, were found to have severe TB as their sole phenotype. Overall, these recent findings provide the proof of concept that the human genetics of TB involves a continuous spectrum from Mendelian to complex predisposition with intermediate major gene involvement. The understanding of the molecular genetic basis of TB will have fundamental immunological and medical implications, in particular for the development of new vaccines and treatments.

[Genetics and immunity of tuberculosis]. / Génétique et immunité de la tuberculose.

Tuberculosis, caused by Mycobacterium tuberculosis, is the most common mycobacterial disease in the world and remains a leading public health problem. Numerous other mycobacterial species are present in the environment and are most often termed atypical or nontuberculous mycobacteria. Like the attenuated vaccine Bacille Calmette-Guérin (BCG) they are generally poorly virulent, even so they can be at the origin of severe infections if the host immune response is impaired. It has been clearly demonstrated that the intrinsic virulence of a mycobacterial species is not the only factor determining disease severity, which is illustrated by the observation that the majority of individuals infected with M. tuberculosis do not develop clinical disease. Numerous arguments suggest that disease severity depends largely on susceptibility/resistance determined by the host genetic make up. In the following review we will discuss the studies on the genes implicated in complex predisposition to tuberculosis and Mendelian predisposition to disease caused by less virulent mycobacteria, proposing a continuous spectrum between those two types of predisposition.

Chronic cases of tuberculosis in Casablanca, Morocco.

OBJECTIVE: To evaluate the prevalence and patterns of drug resistance of Mycobacterium tuberculosis isolates collected from patients with chronic tuberculosis in Casablanca, Morocco. METHODS: Between February 1996 and September 2001, 122 isolates were recovered from 112 different patients. The male to female ratio was 2.4. RESULTS: From February 1996 to May 1997, 77.5% of isolates were multidrug-resistant (MDR-TB), compared to 69.4% from February 1999 to May 2000 and 78.7% from June 2000 to September 2001. The prevalence of MDR-TB is similar from the initial to the last period of this study. Analysis of the 69 bp hypervariable region of the rpoB gene by DNA sequencing on 42 M. tuberculosis isolates (37 resistant, 5 sensitive) showed nine different types of mutations on codons rpoB 513, rpoB 516, rpoB 522, rpoB 523 and rpoB 526. A new point mutation was observed on codon rpoB 523 on one isolate. No mutation was detected on this rpoB region for four resistant isolates. CONCLUSION: The high rate of MDR-TB illustrates a serious problem. The public health authorities have introduced a new regimen protocol consisting of 3 months of kanamycin, ofloxacin, pyrazinamide and ethionamide, followed by 18 months of ofloxacin, pyrazinamide and ethionamide (3KOZEA/18OZEA) for this category of patients, and it is hoped that the additional use of ofloxacin during the intensive phase of treatment will reduce the rate of resistance.

Variants of the human NRAMP1 gene and susceptibility to tuberculosis in Morocco.

It has been well established that the host genetic background is an important modulator of tuberculosis susceptibility. The NRAMP1 (alias SLC11A1) gene has been associated with tuberculosis susceptibility in several ethnic groups. Here we studied the association and linkage of NRAMP1 with tuberculosis in 116 nuclear families, comprising 211 affected offspring, from Casablanca, Morocco. All enrolled tuberculosis cases were culture-positive. No evidence was found of linkage or association of NRAMP1 with tuberculosis. These findings suggest heterogeneity in the genetic control of tuberculosis susceptibility.

Evaluation of IS6110 as amplification target for direct tuberculosis diagnosis.

We describe in the present study an evaluation of the IS6110 repetitive element in the rapid diagnosis of pulmonary and extrapulmonary tuberculosis by polymerase chain reaction (PCR). A pair of oligonucleotide primers was designed to amplify a 201-bp DNA fragment of IS6110. The amplified DNA was detected by ethidium bromide stained agarose gel electrophoresis and confirmed by Sal I digestion and Southern blot hybridization with a 32P-labeled probe. To detect the presence of amplification inhibitors, an internal control DNA that used the same primers as for the target sequence was added to each PCR reaction. PCR results were compared with the results of acid fast stained smears, cultures, and clinical data in 102 sputum and 41 extrapulmonary specimens. With the exception of four samples, M. tuberculosis was detected by PCR in all smear- and culture-positive cases and in all smear-negative, culture positive cases. Additionally, PCR was able to detect 6 cases that were smear and culture negative but clinically strongly suspected of tuberculosis. The final PCR sensitivity and specificity were 93.1% and 95.18%, respectively. One M. tuberculosis strain isolated from a sputum was found to lack IS6110. This study shows that (1) PCR diagnosis based on IS6110 reached the best sensitivity and specificity but must be considered carefully since some M. tuberculosis strains lack IS6110; and (2) PCR must be interpreted in conjunction with clinical and radiological data when it is discordant with conventional methods results.

Survey of primary drug resistance of Mycobacterium tuberculosis in Casablanca, Morocco.

SETTING: In 1990, a 6-month short-course regimen (2 SHRZ/4 RH) was introduced for the treatment of tuberculosis in Morocco. OBJECTIVE: To assess the efficacy of the national tuberculosis control programme, a prospective study of primary drug resistance was conducted from April 1992 to July 1994 in Casablanca. DESIGN: A total of 402 strains isolated from 402 patients living in Casablanca with no previous history of tuberculosis was included in the study. RESULTS: The overall rate of primary drug resistance to at least one drug was 23.9%; it was 19.7% to streptomycin, 11.4% to isoniazid, and 8.2% to both streptomycin and isoniazid. The rates of resistance to rifampicin and ethambutol were both less than 1%. The survey was divided into two periods of 14 months each. The rates of primary drug resistance increased from 21.1% to 27.6% during these two periods (Odds Ratio [OR] 1.43; 95% Confidence Interval [CI] 0.88 to 2.32); this increase occurred only for streptomycin (15.9% to 24.7%, OR 1.73; 95% CI 1.02 to 2.93). CONCLUSION: The rate of primary drug resistance of Mycobacterium tuberculosis in Casablanca has risen in recent years to an ominous level. Urgent measures are needed in order to interrupt this trend.

[Molecular typing of Moroccan strains of Mycobacterium tuberculosis]. / Typage moléculaire de souches marocaines de Mycobacterium tuberculosis.

The insertion sequence IS 6110 was used to differentiate clinical Moroccan isolates of Mycobacterium tuberculosis by using two non radioactive probes. Among 16 strains isolated from patients clinically related, 10 had similar IS 6110 restriction fragment length polymorphism (RFLP) patterns, confirming that they were derived from a common source. Two strains were isolated from the same patient (sputum, lymph node) showed identical profiles hybridized with IS 6110 element. Four sequential strains isolated from the same patients before treatment and after one year had identical IS 6110 RFLP patterns suggesting relapse and not reinfection. Twenty-one strains with identical drug susceptibility showed different IS 6110 RFLP profiles confirming no correlation between antibiotic resistance profiles and IS 6110 RFLP patterns. Since RFLP analysis by using IS 6110 element is a useful tool for the epidemiological survey, of tuberculosis.

[PCR detection of Mycobacterium tuberculosis lacking IS 6110]. / Détection par PCR de Mycobacterium tuberculosis sans IS 6110.

We have evaluated the frequency of M. tuberculosis strains which lack IS 6110 among 102 sputa isolated from Moroccan patients. A pair of primers was designed to amplify a 201bp DNA fragment of IS 6110. The amplified DNA was detected by ethidium bromide stained agarose gel electrophoresis and confirmed by southern blot hybridization with a 32P-labelled probe (PMTO2). To detect the presence of amplification inhibitors, an internal control DNA was added in each negative PCR result. Among 102 samples, 6 sputa were negative by PCR-IS 6110 but culture positive. The test of detection of M. tuberculosis for 2/6 sputa by PCR Amplicor amplifying 584 pb of rRNA 16s sequence was positive. RFLP analysis of these 2 strains revealed no bands hybridizing IS 6110 but PCR-Mt 308 was positive. These results confirmed that these M. tuberculosis strains are lacking IS 6110.