Extensively drug-resistant tuberculosis (XDR-TB) in Morocco

Accepted Manuscript Title: Extensively drug-resistant tuberculosis in Morocco Authors: Wifak Ennassiri, Sanae Jaouhari, Wafa Cherki, Reda Charof, Abdelkarim Filali-Maltouf, Ouafae Lahlou PII: DOI: Reference:

S2213-7165(17)30125-X http://dx.doi.org/doi:10.1016/j.jgar.2017.07.002 JGAR 449

To appear in: Received date: Revised date: Accepted date:

7-3-2017 25-5-2017 12-7-2017

Please cite this article as: Wifak Ennassiri, Sanae Jaouhari, Wafa Cherki, Reda Charof, Abdelkarim Filali-Maltouf, Ouafae Lahlou, Extensively drug-resistant tuberculosis in Morocco (2010), http://dx.doi.org/10.1016/j.jgar.2017.07.002 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Extensively Drug-Resistant Tuberculosis in Morocco

Wifak Ennassiria, b, Sanae Jaouharib, Wafa Cherkib, Reda Charofb, Abdelkarim Filali-Maltoufa, Ouafae Lahloub*

a

Laboratory of Microbiology and Molecular Biology, Faculty of Sciences, Mohammed V

University, Rabat, Morocco b

National Tuberculosis Reference Laboratory, National Institute of Hygiene, Rabat, Morocco

*Corresponding author: Ouafae Lahlou Postal address: Laboratoire National de Référence de la Tuberculose, Institut National d’Hygiène, 27 Avenue Ibn Batouta, BP 769, Rabat, Maroc E-mail address: [email protected] Phone number: 00212 661 823 342

Fax: 00212 537 686 192

Highlights  Drug-resistant tuberculosis threatens the TB control efforts in Morocco.  This is the first study to detect XDR M. tuberculosis strains in Morocco. Detection of the drug resistance associated mutations using the GenoType MTBDRsl assay.

Abstract Objectives: Extensively drug-resistant tuberculosis (XDR-TB) has recently been identified as a major global health threat. The aim of the study was to evaluate the XDR-TB presence within TB isolates in Morocco, and its association with the demographic, clinical and epidemiological features. 1

Methods: A total of 524 patients from the National Tuberculosis Reference Laboratory, representative of all the geographic regions, were subject to first-line drug susceptibility testing (DST). 155 isolates found to be multidrug-resistant tuberculosis (MDR-TB) underwent secondline DST. Moreover, to enhance our understanding of the genetic basis of these drug-resistant strains, we investigated drug resistance associated mutations in isolates either identified as preXDR and XDR or suspected resistant using the GenoType® MTBDRsl V1.0 assay. Results: In this study, we identified 4 (2.6%) XDR and 18 (11.8%) pre-XDR isolates. The agreement between the MTBDRsl assay results and the phenotypic DST was 95.2% for Ofx, 81% for Km and 95.2 % for Amk. Conclusion: To the best of our knowledge, this is the first study to evaluate the frequency of XDR M. tuberculosis strains in Morocco. Our result highlights the need to reinforce the TB management policy in Morocco with regard to control and detection strategies to prevent further spread of XDR isolates.

Keywords: Mycobacterium tuberculosis, MDR-TB, XDR-TB, GenoType MTBDRsl, Morocco.

1. Introduction Mycobacterium tuberculosis is the causative agent of tuberculosis (TB). Despite efforts mobilized in the context of TB control, the disease continues to be a major global health problem worldwide, with an estimated 10.4 million new TB cases and 1.4 million deaths from the disease in 2015 [1]. 95% of the deaths occur in low and middle-income countries [2]. Increasing cases of drug-resistant M. tuberculosis strains continue to emerge and have become urgent and alarming threats worldwide [3]. The multidrug-resistant tuberculosis (MDR-TB), denoting resistance to both rifampicin (RMP) and isoniazid (INH), and extensively drug-resistant tuberculosis (XDR-TB) characterized as MDR strains additionally resistant to fluoroquinolones 2

and to at least one of the three second-line injectable drugs, has increased the threat [4,5]. Drugresistant tuberculosis (DR-TB) is either acquired due to spontaneous genetic mutations, efflux mechanisms, malabsorption or transmission from infectious DR-TB patients. Further, the inadequate dosing and poor adherence to the therapeutic regimen in TB patients generates and fuels the development and the rise of MDR and XDR-TB [6-8]. Even though there are improvements in TB treatment, leading to a 83% success rate among all new TB cases, only 52% of MDR-TB patients are treated successfully [1,9]. Globally, an estimated 3.9% of new cases have MDR-TB; the proportion is higher among previously treated cases, at 21%. In 2015, there were an estimated 480 000 new cases of MDR-TB worldwide, and approximately 250 000 deaths from rifampicin-resistant and MDR-TB [1,9]. Also, some MDR strains have additional drug resistance, such as pyrazinamide and/or streptomycin that might decrease treatment efficiency [10]. The first case of XDR-TB was notified in South Africa in 2005 [11]; by the end of 2015, 117 countries had reported at least one case of XDR-TB. The average proportion of MDR-TB cases with XDR-TB was 9.5%, while the proportion of MDR-TB cases with resistance to any fluoroquinolone, including ofloxacin, levofloxacin and moxifloxacin, was 21% [1,3]. In Morocco, according to World Health Organization (WHO) estimates for the year 2015, the annual number of incident cases of TB was approximately 37 000 cases, with a mean incidence rate of 107 cases per 100 000 inhabitants [12]. On the other hand, in the 2015 report released by Morocco’s National Tuberculosis Control Program (NTCP), there were a total of 30 636 cases; of which 48% had the pulmonary tuberculosis form; and an incidence of 89 cases per 100 000 inhabitants, has been recorded [13]. Moreover, according to the 2014 Moroccan national primary and secondary resistance to anti-tuberculosis drugs survey based on the WHO protocol, the prevalence of MDR-TB among new and retreatment cases was 1% and 8.7% respectively [12,13]. 3

Despite the fact that there is a low resistance rate detected in Morocco, the management of MDRTB poses a problem. Indeed, the intrafamilial transmission of tuberculosis, in addition to the low treatment success rates, are still threatening the TB control efforts. Knowledge of M. tuberculosis isolates resistance patterns is important for the effective clinical management of TB patients and for the control of the disease. Thus, this investigation aims to evaluate the presence of the XDR M. tuberculosis in Moroccan patients and to determine the associated demographic, clinical and epidemiological features in favor of generating XDR-TB.

2. Materials and methods In resource-limited countries, WHO guidelines recommend the TB diagnosis by smear microscopy in all new TB cases, and by smear microscopy, culture, and phenotypic and genotypic drug susceptibility testing (DST) in both retreatment cases and DR-TB suspected cases [14-16].

2.1. Study population During 2015, a total of 155 isolates were identified as MDR by DST from a panel of 524 strains sent to the National Tuberculosis Reference Laboratory (NTRL) in the National Institute of Hygiene (NIH), Morocco; at the clinician’s request. These strains were obtained from the sputum of drug-resistant suspected patients representing all the geographic regions of Morocco and belonged to TB patients with different therapeutic backgrounds, either treatment failure using the first-line drugs, TB relapse cases, or MDR-TB contact subjects.

4

Note that sampling and testing for all the isolates were performed as part of routine clinical practice for TB diagnosis and drug resistance detection, according to the NTCP guidelines, and no sampling or investigations were oriented to research needs. At the time of the collection of samples, all the patients were aware that their samples will be used for TB diagnosis and drug resistance detection, for their benefit. Ethical approval and informed consent were, thus, not required. Patients’ data were collected using a test inquiry form as is normal procedure during the clinical consultation process. The treatment for these MDR-TB patients was based on the 2011 NTCP recommended standardized treatment, which is consistent with the WHO recommendations [17,18]. However, according to the collected patients data the 4 major treatment regimens prescribed were: 8 months Km-Ofx-Eto-Z-E/16 months Ofx-Eto-Z-E; 8 months Km-Lfx-Cs-Eto-Z/16 months Ofx-Cs-Eto-Z; 8 months Cm-Lfx-Cs-Eto-PAS-Z/16 months Lfx-Cs-Eto-PAS-Z and the 8 months Cm-High dose Lfx -Cs-PAS-Z/16 months Lfx-Cs-PAS-Z. Demographic and clinical data has shown that the majority were males (71%), and most were between 15 and 72 years old (Table 1).

2.2. Strains isolation and drug susceptibility testing All the specimens were decontaminated and liquefied with the petroff method; the inoculation was performed on Lowenstein-Jensen (L/J). If cultures were positive and demonstrating growth of M. tuberculosis, the isolates were then tested for drug resistance. Growth confirmation included a macroscopic analysis of colonies on L/J medium and a microscopic analysis. The DST was done as a two stage process, initially for first-line drugs then, for second-line drugs for the MDR isolates. The tested first-line drugs are: isoniazid, rifampicin, ethambutol, and streptomycin, and the test was performed using the proportion method according to the WHO guidelines [14,19,20], 5

with the following critical drug concentrations : RMP, 40µg/ml; INH, 0.2 µg/ml; SM, 4 µg/ml and EMB, 2 µg/ml. The strains presenting multidrug resistance were subject to second-line drugs DST for ofloxacin (Ofx), kanamycin (Km), and amikacin (Amk); the drugs critical concentrations in the medium are respectively: 2 µg/ml, 30 µg/ml, 40 µg/ml. The M. tuberculosis H37Rv was used as a quality control testing in DST. The tubes were examined for bacterial growth after 28 days of incubation. If no growth or little growth was observed, the tubes were left for 42 days. The strain was defined as resistant when ≥1% of the M. tuberculosis population grows in the presence of the critical concentration of the drugs [19]. The NTRL is evaluated annually in phenotypic and genotypic first and second-line DST by the TB Supranational Reference Laboratory at the Institute of Tropical Medicine, Antwerp, Belgium.

2.3. GenoType MTBDRsl V1.0 The GenoType® MTBDRsl V1.0 assay developed by Hain Lifescience, Nehren, Germany, is one of the methods widely used for the identification of mutations linked to M. tuberculosis drug resistance to fluoroquinolones (FQs) and second-line injectable drugs (SLIDs) [21]. In order to evaluate the usefulness and diagnostic accuracy of the assay, 21 isolates either showing or suspected to have resistance to any of the second-line drugs using conventional DST, were selected. The MTBDRsl assay was performed on isolates after solid culture. Briefly, the test was processed as follows: Genomic DNA was extracted using the GenoLyse® kit (Hain Lifescience, Nehren, Germany), followed by multiplex polymerase chain reaction amplification (PCR), and finally the reverse hybridization was performed as per manufacturer’s instructions. A test is valid and interpretable, when conjugate controls (CC), amplification control (AC) and M. tuberculosis complex (TUB) bands are visible along with the genes locus control (LC); absence of a wild-type band or the presence of a mutant band was indicative of a resistant isolate.

6

3. Results 3.1. Patients This study included 155 patients with clinical, radiological, and bacteriological evidences of MDR-TB. The 155 M. tuberculosis isolates originated from patients living in cities located all across the country. The demographic and epidemiological data summarized in Table 1 were obtained from laboratory records. Overall, males represented 71% (110/155) of the cases, with a male to female sex-ratio of 2.4 (110/45) and an age range of 15 to 72 years. Regarding their clinical status, 21.9 % (34/155) of the patients were new cases and 78.1% (121/155) were previously treated. Data regarding substance dependence, and the HIV serological status was not available for all the patients. However, from the reported information only one patient was HIV positive 1.1% (1/91), 98.9% (90/91) were HIV negative. At the time of the diagnosis, 15.4% (14/91) were smokers, 9.9% (9/91) were both smoking and using drugs or alcohol, 74.7% (68/91) were former smokers or had never smoked. The patients’ monitoring showed that among the 155 patients, 8 were declared cured, 46 showed negative culture, 2 patients were transferred outside the country, 7 patients were lost to follow-up treatment, 19 were still showing a positive culture, whereas 8 patients died. 65 patients are undergoing treatment. 3.2. Drug-susceptibility patterns The second-line DST data was available for all except 2 patients (n=153); among these obtained MDR strains; 85.6% (131/153) were second-line sensitive and 2.6% (4/153) strains were found to be XDR. Of the remaining 18 strains, 9.8% (15/153) presented resistance to Ofx, 1.3% (2/153) were resistant to Km, and 0.7% (1/153) presented resistance to both Km-Amk. These 18 strains were categorized as pre-XDR (Table 2).

7

3.3. Analysis of the GenoType MTBDRsl results for detecting resistance to Ofx, Km, and Amk The agreement between the MTBDRsl assay and the phenotypic DST results for the 21 isolates, subjected to the genotypic test summarized in Table 3 was 95.2% for Ofx, 81% for Km and 95.2 % for Amk. Indeed, some discrepancies between the phenotypic DST and the MTBDRsl results were apparent for 6 strains. These discrepancies were observed in both pre-XDR and XDR strains. 5 strains (3 pre-XDR and 2 XDR strains) of which showed no mutations in the genotypic test and were identified as phenotypically resistant, 1 for Ofx and 4 for Km. The remaining strain (XDR strain) which was found phenotypically susceptible to Amk was identified as resistant by the genotypic assay. The distribution of the genotypic patterns and mutations in the 16 FQ and 2 aminoglycosides/cyclic peptid (AG/CP) resistant isolates identified by the MTBDRsl assay are shown in Table 4. The predominant mutations identified as conferring FQs resistance were gyrA MUT3C (D94G) (37.5%), gyrA MUT1 (A90V) (25%), gyrA MUT3B (D94N/D94Y), gyrA MUT3D (D94H), and missing WT1 (6.25%). Dual mutations gyrA MUT1+MUT3C (A90VD94G), gyrA MUT3B+MUT3D (D94N/D94Y- D94H) and missing gyrA WT2+gyrA MUT1, were identified in 3 isolates. Using the MTBDRsl assay, the A1401G mutation in the rrs gene was identified in 1 isolate. The other isolate showed an absence of the rrs WT2. 4. Discussion Similar to many other developing countries, DR-TB is increasing in Morocco. It is important to note that a high proportion of drug resistance cases among young age groups is more likely to be indicative of recent transmission, in older age groups, it’s more likely to be an indicator of reactivation of old infections [22]. Actually, the TB patients whose MDR isolates were analyzed in this study were predominantly young adults in their most productive years; 62.41% of the cases were in the age group of 15–44 years old and the median age was 35.8 (Table1); 8

therefore it has a high impact on the socio-economic status of the country, and a higher transmission risk. Other studies in Morocco showed a similar age distribution among TB patients [23,24]. The findings in our study show that among these MDR-TB cases, 4 cases (2.6%) were further determined to be XDR-TB, while 18 (11.8%) were pre-XDR-TB (Table 2). However, the proportion of XDR-TB among the MDR-TB cases from our study (2.6%) seems low compared to the international scale. As a matter of fact, such a difference might be the result of the low proportion of MDR-TB in Morocco in comparison with the continuous surveillance or surveys data reported by other countries recording over ≥10% of XDR-TB cases such as Belarus, Lithuania, Latvia and Georgia. Additionally, in the study carried out by Gandhi et al., 23.98 % (53/221) of MDR-TB cases in South Africa were detected as XDR-TB in the KwaZulu-Natal, a province registering high incidence of MDR-TB [11]. Another interesting observation was that all of the 22 pre-XDR and XDR-TB patients were HIVnegative in the present study. This finding is in agreement with Sharma SK and al. study that had reported that HIV infection is more common among the general population than in DR-TB patients [25]. Also, patients of both genders carried drug-resistant TB isolates, and all age groups were at risk; the age of our pre-XDR and XDR-TB patients varied between 22 and 60. The geographic distribution showed that; patients originated from different regions of Morocco. Hence, no association neither to sex, age nor origin could be established. Regarding the category of patients; one of the 22 patients was diagnosed as having primary pre-XDR-TB, with no history of previous treatment and no known close contact whereas all the remaining 21 patients had a previous antiTB treatment history. As a result we conclude that the risk for developing pre-XDR and XDRTB is much higher in those previously treated. This is in agreement with the national primary and secondary resistance to anti-tuberculosis drugs survey results, in which it is reported that the MDR-TB prevalence in Morocco among retreatment is higher than among new cases [1,12]. 9

Our study has shown that out of the 18 pre-XDR isolates, 15 were Ofx resistant. Indeed, the inappropriate use of drugs, may lead to occurrence of these pre-XDR isolates especially FQ resistance [21]; this might be explained by the fact that the FQs, in particular Ofx, is the most commonly prescribed antibiotic for respiratory tract infection, acute otitis externa, urinary infections and intestinal infections. Since the M. tuberculosis drug resistance is due to the gradual accumulation of mutations in the genome, these pre-XDR isolates are always at the risk of developing XDR-TB [26]. In an attempt to better understand the genetic basis of DR-TB, we investigated drug resistance associated mutations, by performing the MTBDRsl assay on 21 pre-XDR, XDR and suspected resistant isolates. The study results showed that inconsistency existed between the genotypic and phenotypic DST results (Table 3), proving that the molecular diagnosis may be used as an initial test for second-line drug resistance. However, the wild-type results yielded by the assay should be confirmed by phenotypic DST. Actually, the assay was developed with a specific focus on the most prevalent gyrA, rrs gene mutations; only mutations covered by either wild-type or mutant probes can be detected by the assay. Novel mutations and genetic changes, besides in the targeted regions, can lead to misinterpretation and create a discrepancy in results, which is in agreement with the observations described by our study [21,27]. The M. tuberculosis FQs resistance has been predominantly attributed to gyrA mutations. A study by Li J and colleagues suggested that isolates with the D94G and D94N mutations were associated with a high resistance level, whereas isolates carrying the A90V and D94A mutations were associated with a low resistance level [28]. In this study, we found high mutation frequencies in codon 94 followed by codon 90. As reported in Table 4 and considering the three isolates which carried dual mutations 10/16 (62.5%) isolates presented a high level resistance, while 6/16 (37.5%) presented a low level resistance. Furthermore, various reports have linked the rrs gene mutations A1401G, C1402T, and G1484T to Amk, Cm, and Km resistance, associating them with specific 10

resistance levels. Mutations G1484T and A1401G were reported to cause high-level resistance to all drugs, whereas C1402T causes resistance to only Cm and Km [21]. Our study included one single isolate with a confirmed A1401G mutation which was Km and Amk resistant, while the other isolate showing an absence of the rrs WT2 associated with the codon 1484, identified as Km resistant and Amk susceptible. An explanation might be that the proportion of predominant wildtype bacteria masks the presence of the Amk resistant organisms in the phenotypic DST. These two isolates were classified as high level resistant. The patients were monitored to assess their response to therapy. Among the 46 patients showing negative culture only 2 belonged to pre-XDR-TB patients’ category. Based on these findings, we conclude that the median time to conversion from a positive to a negative culture is longer for patients with XDR-TB than for patients with MDR-TB. Compared to patients with either drugsusceptible TB or MDR-TB infections, XDR-TB infected patients have poorer outcomes. Furthermore, high mortality risks have been reported for XDR-TB patients; reaching 50%, compared to those with MDR-TB infections [29]. Only one death was reported among the preXDR-TB patients in our country. The 7 remaining cases of death that occurred were carrying second-line susceptible isolates and these cases show that both patients and physicians are playing significant roles in the prevention and in the treatment of MDR and XDR-TB diseases, since these infections are too often the result of patients’ noncompliance with treatment regimens [30]. There have been few recommendations for the treatment of XDR-TB, and amongst these, results of observational studies have shown that even though the MDR and XDR-TB treatment has few viable or effective solutions, it can be successful for some patients [31]. Indeed, according to the WHO, with the conventional regimen, 52% of MDR-TB patients are treated successfully worldwide [1,9]. However, the estimated treatment success among the rifampicin resistant and MDR-TB patients in Morocco is solely 42% [12]. The NTCP reported that the MDR-TB healing rate ranges between 30% and 40% [13]. Recently, data of observational studies from patients 11

treated using shorter regimens, of 9 months instead of 24 months, in African and Asian locations have shown much higher likelihood of treatment success compared with longer conventional regimens [32]; treatment success was reported in 84% of these patients. Regarding the XDR-TB treatment success rates, several countries with the appropriate management of drug resistance programs have shown that recovery is possible for 30% to 50% of XDR-TB affected people. However, the success of the treatment depends greatly on the extent and degree of the drug resistance, the severity of the disease and whether the patient's immune system is weakened [33, 34]. To ensure successful outcomes, second-line drugs must be used judiciously and cautiously by the physicians, and the strict monitoring of treatment is essential [35]. Therefore the WHO's approach is the prompt implementation of DOTS to reduce the number of TB deaths, infections and the chances for drug resistance development [31,36]. The use of sputum smear microscopy and culture rather than sputum smear microscopy alone is recommended for the monitoring of patients with MDR-TB and XDR-TB during treatment [32]. Following the identification of MDR, pre-XDR and XDR strains in Morocco, the NTCPs technical committee held a series of meetings to develop both a plan and a guide of concrete actions to support the MDR and XDR-TB, taking into account the WHO 2016 new recommendations, DRTB treatment guidelines and the WHO's new End TB Strategy 2016–2035 [32,37]. Indeed, the NTCP fervently works at the national level for the elimination of the anarchic treatments and the implementation of the newly national DR-TB diagnostic algorithm including the integration of new genotypic laboratory drug susceptibility testing methods [15,16,37] and the shorter regimens to increase the chances of successful treatment and improved adherence.

5. Conclusion

12

The XDR-TB has a high mortality rate and causes concern in TB management due to a sharp decline in therapeutic possibilities. The present study was an attempt to evaluate the frequency of XDR-TB cases. Actually, future large-scale prospective screening studies and drug resistance surveillance procedure, along with a characterization of the genetic diversity, population structure and tuberculosis transmission dynamics in Morocco, will be conducted. For this purpose highresolution molecular typing, such as spoligotyping, MIRU-VNTR typing and gene sequencing methods will be used, which may help us to establish new management policies of DR-TB. Indeed, increased efforts should be made to ensure that all patients diagnosed with MDR-TB undergo testing for susceptibility to FQs and SLIDs. Surely, this will be of great benefit in adapting treatment regimens, limiting both the dissemination of MDR strains, and the emergence of XDR strains in Morocco. Acknowledgments We are sincerely grateful to Pr. Veronique Vincent the TB laboratory expert and Pr. Jamal Eddine Bourkadi specialist in pneumo-phtisiology and director of the university health center (Moulay Youssef Hospital) for their thoughtful reading and evaluation of the manuscript.

Funding: This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Conflicts of interest: The authors declare that they have no conflict of interest. Ethical approval and informed consent: This work was undertaken as part of routine clinical practice for TB diagnosis according to the NTCP guidelines. Ethical approval and informed consent were, thus, not required.

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Table 1. Demographic and epidemiological data of the studied population from Morocco (n=155) Previously Administrative regions

Sex-

New Cases Mean age

(cities)

ratioa

Total Nb treated cases Nb

Nb (%)

of Cases (%)

Tanger-Tetouan-Al Hoceima

2

37.75

4 (44.4)

5 (55.6)

9

Casablanca-Settat

1.75

36

5 (22.7)

17 (77.3)

22

Fès-Meknès

2.1

34

2 (6.4)

29 (93.6)

31

Rabat-Salé-Kénitra

3.11

34.44

15 (21.4)

55 (78.6)

70

Souss-Massa

2.66

46.6

4 (36.4)

7 (63.6)

11

Marrakech-Safi

2.5

41

3 (42.9)

4 (57.1)

7

L’oriental

NAb

NA

0

1 (100)

1

1

34.75

1 (25)

3 (75)

4

2.44

35.8

34

121

155

Beni Mellal-Khénifra Total a Male to Female sex ratio.

b NA: Not Applicable since only 1 patient originated from this region.

18

Table 2. Antimicrobial susceptibility profile among pre-XDR-TB and XDR-TB isolates by phenotypic DST (n=22). No. Of INH

RMP

SM

EMB

OFX

KM

AMK

4

R

R

S

S

R

S

S

2

R

R

R

S

R

S

S

9

R

R

R

R

R

S

S

1

R

R

R

S

S

R

S

1

R

R

R

R

S

R

S

1

R

R

S

S

S

R

R

2

R

R

S

S

R

R

S

1

R

R

R

S

R

R

R

1

R

R

R

R

R

R

S

strains

R : resistant; S : susceptible; RMP : Rifampicin; INH : Isoniazid; SM : Streptomycin; EMB : Ethambutol; OFX : Ofloxacin; KM : Kanamycin; AMK : Amikacin. The strains in the last lines (bold) are classified, per definition, as XDR-TB.

19

Table 3. Comparison of the results obtained by the GenoType MTBDRsl assay and phenotypic testing for: Ofx, Km, and Amk. No. Of isolates with the indicated results by phenotypic DST/MTBDRsl assay Drug Agreement

Discrepancy

R/R

S/S

R/S

S/R

Ofloxacin

16

4

1

0

Kanamycin

2

15

4

0

Amikacin

1

19

0

1

20

Table 4. GenoType MTBDRsl patterns and mutations detected gyrA and rrs MTBDRsl Pattern and mutation

No. Of DST results

Codon mutation

results

% isolates

OfxR (n=16) FLQR

OfxR

A90V

4

25

FLQR

OfxR

A90V

1

6.25

gyrA MUT3B

FLQR

OfxR

D94N/D94Y

1

6.25

gyrA MUT3C

FLQR

OfxR

D94G

6

37.5

gyrA MUT3D

FLQR

OfxR

D94H

1

6.25

FLQR

OfxR

A90V and D94G

1

6.25

FLQR

OfxR

1

6.25

1

6.25

gyrA MUT1 ∆gyrA WT2+gyrA MUT1

gyrA MUT1+MUT3C gyrA

D94N/D94Y and

MUT3B+MUT3D

D94H

∆gyrA WT1

FLQR

OfxR

∆WT1b

WTa

FLQS

OfxR

1

WT

FLQS

OfxS

4

rrs MUT1

AG/CPR

KmR, AmkR

A1401G

1

50

∆rrs WT2

AG/CPR

KmR, AmkS

∆WT2

1

50

WT

AG/CPS

KmR, AmkS

4

WT

AG/CPS

KmS, AmkS

15

KmR (n=2)

a WT, wild-type. b ∆WT, lack of hybridization with the indicated wild-type probe

21