Lucy OBONYO NYANG'AU

Arch Microbiol Immunology 2020; 4 (2): 57-65 10.26502/ami.93650045
Archives of Microbiology & Immunology Vol. 4 No. 2 – June 2020 57
Research Article
False Positive Tuberculosis Cases (Xpert MTB/RIF Assay)
Among People Living With HIV Attending Bahati
Comprehensive Care Centre Nairobi, Kenya
Lucy Obonyo Nyang’au1*, Evans Amukoye2, Stanley Kangethe3, Jackson Onyuka4
1Mount Kenya University, Thika, Nairobi, Kenya
2Kenya Medical Research Institute, Nairobi, Kenya
3Mount Kenya University, Thika, Nairobi, Kenya
4Mount Kenya University, Thika, Nairobi, Kenya
*Corresponding Author: Lucy Obonyo Nyang’au, Department of Medical Laboratory Sciences, Mount
Kenya University, P.O Box 342-01000, Thika, Kenya; Tel: +254-0722816265; E-mail:
lucynyangau@yahoo.com
Received: 06 April 2020; Accepted: 16 April 2020; Published: 24 April 2020
Citation: Lucy Obonyo Nyang’au, Evans Amukoye, Stanley Kangethe, Jackson Onyuka. False Positive
Tuberculosis Cases (Xpert MTB/RIF Assay) Among People Living With HIV Attending Bahati Comprehensive
Care Centre Nairobi, Kenya. Archives of Microbiology & Immunology 4 (2020): 57-65.
Abstract
The introduction of GeneXpert MTB/RIF assay has
impacted positively in tuberculosis diagnosis,
providing a rapid way of identifying tuberculosis
patients in high burden, low income countries.
However Mycobacterium tuberculosis (MTB)
detection in previously treated patients, which may
be due to old deoxyribonucleic acid or active
disease, still remains a diagnostic dilemma for
diagnosis of tuberculosis. A retrospective cohort
study was conducted among consenting patients
with signs and symptoms of tuberculosis attending
Bahati comprehensive care centers. A total of three
hundred and forty six patients were sampled and
their sputa collected, subsequently laboratory
analysis was carried out for detection and culture of
Mycobacterium tuberculosis. Seventy seven (22%)
sputa had Mycobacterium tuberculosis detected on
Xpert MTB/RIF assay sputa from these patients
with bacteriologically confirmed pulmonary
tuberculosis were subjected to culture on
Mycobacterium Growth Indicator Tube (MGIT)
media. Detection of Mycobacterium tuberculosis
on Xpert MTB/RIF assay with no isolation of
growth on culture indicated a false positive
tuberculosis diagnosis. Out of 77 isolates subjected
for culture a total of 0(0%) and 5(7.5%); P=0.484,
isoniazid preventive therapy and non- isoniazid
preventive therapy patients had false positive
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Archives of Microbiology & Immunology Vol. 4 No. 2 – June 2020 58
tuberculosis cases, while 0(0%) and 5(25%);
P=0.001 new and retreatment patient’s had false
positive tuberculosis. Our study concluded that
there was no significant association between
isoniazid preventive therapy and tuberculosis false
positivity but there was significant association
between patient treatment status and tuberculosis
false positivity. Previously treated tuberculosis
patients were significantly associated with false
positivity, this call for clinicians to exercise caution
when interpreting results from previously treated
tuberculosis patients.
Keywords: False positive tuberculosis;
GeneXpert MTB/RIF assay; Mycobacterium
tuberculosis
1. Introduction
Tuberculosis is one of the top ten causes of death
globally and the leading cause of death from a
single infectious agent (ranking above HIV/AIDS)
[1]. Despite declining global incidence and
mortality tuberculosis (TB) remains a major
challenge worldwide, an estimated 10.0 million
people fell ill with TB in 2018, with estimated 1.2
million TB deaths among HIV negative people and
an additional 2,51,000 deaths among HIV positive
people were recorded respectively [1]. TB affects
people of all age groups and gender, the highest
burden was in Men aged >15 years in 2018, they
accounted for 57% of all TB cases, while Women
accounted for 32% and children aged < 15 years
accounted for 11% TB cases globally [1].
People living with HIV (PLHIV) accounted for
8.6% TB cases in 2018 [1]. HIV co-infection is
associated with unusual presentations of TB such
as smear negative and abnormal chest radiographs
this causes a diagnostic challenge, poor treatment
outcome and subsequent increased mortality [2].
Previous studies have shown that pauci-bacillary
forms of TB are more commonly identified in
patients who are HIV positive and these patients
happen to be sputum smear negative, but because
microscopy is less sensitive in these populations
these groups are the ones most likely to benefit
from Xpert MTB/RIF assay [2].
The introduction of GeneXpert MTB/RIF assay has
contributed a huge positive impact in tuberculosis
(TB) diagnosis, by providing a rapid way of
identifying TB patients in high burden, low income
countries [3]. The Xpert MTB/RIF assay is an
automated cartridge based nucleic acid
amplification test (NAAT) capable of
simultaneously detecting Mycobacterium
tuberculosis complex (MTBC) and Rifampicin
(RIF) resistance within 2 hours; this assay was
endorsed by the WHO in 2010, and approved by
FDA in 2013, and it is regarded as a breakthrough
in TB diagnostics [3]. The assay is performed on
the Cepheid GeneXpert multi-disease instrument
system which integrates sample purification,
nucleic acid amplification, and detection of target
sequences [3]. It uses hemi-nested real time PCR
(polymerase chain reaction) for the detection of
MTBC specific sequence of the rpoB gene and five
molecular probes to detect mutations within the
genes rifampicin resistance determining region
(RRDR). The assay can be performed directly on
raw sputum or concentrated sediments.
Nevertheless post implementation studies have
identified several challenges [4], emphasizing the
need for deeper understanding of clinical and
operational factors affecting performance.
2 Materials and Methods
Following approval by the ethics review committee
of Mount Kenya University (Ref.
No.MKU/ERC/1305) and research clearance by
National Commission for Science Technology and
Innovation (NACOSTI/P/19/13045/31000), 346
respondents were recruited for the study using
cluster random sampling.
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Archives of Microbiology & Immunology Vol. 4 No. 2 – June 2020 59
2.1 Study Design
Retrospective cohort study design was used where
eligible HIV positive participants with or without
use of isoniazid preventive therapy (IPT) were
recruited through cluster random sampling, only
those who gave consent were enrolled in the study.
2.2 Study Site
The study was conducted in Makadara sub-county,
Nairobi the capital city of Kenya. The sub-county
covers an area of 13Km2 and comprises of five
wards; Maringo, Hamza, Viwandani, Harambee,
and Makongeni [5]. Viwandani ward is an informal
settlement which is characterized by increased
population. Tuberculosis being airborne,
congestion especially in the houses facilitates
increases in tuberculosis transmission.
2.3 Sample Size Determination
Data from National tuberculosis, leprosy and Lung
disease program (NTLD-P) annual report (2017)
for Nairobi county, a TB prevalence of 0.1%
(147per 100,000) in HIV positive people was
established. Using Habib et al., (2014) [6] formula
and 0.1% as the working prevalence rate (P) for
tuberculosis and assuming a standard error (Z)
from the mean of 1.96 and in absolute precision (d)
of 5%, and design effect (D) being taken as (2)
sample size (n) was calculated as follows.
(1.96)2 (0.1) (0.9)/ (0.05)2 = 276 Clients
The estimated sample size was 276; it was adjusted
to allow for attrition /refusals which was estimated
for 20% [7] thus n= 276/ (1-0.2) =346 Clients.
2.4 Inclusion and Exclusion Criteria
Patients who were HIV positive, on care, above 15
years, with signs and symptoms of tuberculosis,
one year post isoniazid preventive therapy (IPT),
were included in the study upon consent. While
patients with isoniazid preventive therapy and age
records not clear, less than one year post isoniazid
preventive therapy, unable to consent, with other
samples other than sputum were excluded from the
study.
2.5 Laboratory Methods
2.5.1 Identification of Mycobacterium
tuberculosis
Using the geneXpert MTB/RIF assay sputa and the
reagent buffer were mixed according to the
standard operating procedure and loaded into the
Xpert MTB/RIF assay cartridge and test started on
Xpert MTB/RIF assay machine platform [8].
2.6 Culture of Mycobacterium tuberculosis
Mycobacterium tuberculosis (MTB) culture was
performed using non-radiometric method
Mycobacterium Growth Indicator Tube (MGIT)
BACTEC 960. Sputa decontamination was
performed using sodium hydroxide solution (40%
w/v) combined with 2.9% sodium citrate solution
and N-acetyl-L-cystein (NALC) powder [9]. Sterile
phosphate buffer was added and the organisms
concentrated by centrifugation at 3,000 rpm for 15
minutes. The supernatant was decanted and the
sediment suspended with phosphate buffer and
inoculated in liquid MGIT media and incubated
along with negative control (un-inoculated MGIT
media) and positive control (H37Rv ATCC 27294)
[9]. The MGIT tubes were incubated in the
BACTEC MGIT 960 machine at 37oC until the
instrument flagged them positive. After a
maximum of six weeks the instrument flagged the
tubes negative, if there was no growth at 37oC [10-
13]. Confirmative identification of MTB was done
using BD MGIT TBc, on all positive cultures.
Positive culture for MTB confirmed diagnosis of
active disease.
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3. Data management and Statistical
Analysis
Data was tabulated in a computer database
designed using MS-Access, and then transferred to
statistical package for the social sciences (SPSS)
version 20.0 for analysis. Pearson’s Chi-square test
was applied to determine the differences in
proportion for both groups in isoniazid preventive
therapy (IPT) status, type of patient and gender
against the detection of Mycobacterium
tuberculosis (MTB). Pearson’s Chi-square test was
applied to determine the differences in proportion
for both groups in isoniazid preventive therapy
(IPT) status, type of patients and demographics
against TB and TB false positives. While Fisher’s
exact test was applied to determine the difference
in proportions among the MTB detection levels in
GeneXpert MTB/RIF assay and among the age
groups. These results were presented by
appropriate tabulations based on the determined
variables, odds ratio (OR) with 95% confidence
interval (CI) and the corresponding p values. The
threshold for statistical significance was set at P≤
0.05.
4. Results
Prevalence of Tuberculosis false positives in
relation to Isoniazid Preventive Therapy and
Treatment status
Of the 77 Mycobacterium Tuberculosis (MTB)
positive Xpert MTB/RIF assay samples subjected
to culture 5(7.5%) and 0(0%) were false positive
for tuberculosis (TB) among the non-isoniazid
preventive therapy and isoniazid preventive therapy
arms respectively, P=0.484 while on the other hand
0(0%) and 5(25%) were false positive for TB
among new and retreatment patients P= 0.001
(Table 1). This indicates that there was no
significant association between isoniazid
preventive therapy and TB false positivity, while
on the other hand there was significant association
between TB false positivity and retreatment
patients. Further study findings indicated that
10(100%) and 62(92.5%) were true Xpert
MTB/RIF assay TB results in isoniazid preventive
therapy and Non- isoniazid preventive therapy
patients respectively (Table 1). While 57(100%)
and 15(75%) were true Xpert MTB/RIF assay TB
results among new and retreatment TB patients
respectively (Table 1). The true results were
concordant in Xpert MTB/RIF assay and BACTEC
MGIT 960 culture results while the false positive
cases had discordant results in the two methods.
Culture was taken as the reference standard.
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Table 1: False positive tuberculosis cases among study patients
Variables Total (N) True Xpert results n (%) False positives n (%) OR (95% CI) P Value
MTB+ve/Growth MTB+ve /No Growth
IPT status
IPT patients 10 10 (100) 0 (0) UD
0.484
Non-IPT patients 67 62 (92.5) 5 (7.5)
Type of patients
New patients 57 57 (100) 0 (0) UD
0.001
RT patients 20 15 (75) 5 (25)
MTB detection
levels
MTB detected
high
26 25 (96.2) 1 (3.8)
MTB detected
medium
45 41 (91.1) 4 (8.9) 0.690 (0.205-
1.226)
0.999
MTB detected
low
4 4 (100) 0 (0) UD 0.895
MTB detected
very low
2 2(100) 0 (0) UD 0.999
Gender
Female 30 28 (93.3) 2 (6.7) 0.92 (0.144-
5.867)
0.652
Male 47 44 (93.6) 3 (6.4)
Age (years)
< 20 4 4 (100) 0 (0)
20 – 39 47 42 (89.4) 5 (10.6) 0.111 (0.09-
1.271)
0.388
40 – 59 23 23 (100) 0 (0) UD 0.999
60 + 3 3 (100) 0 (0) UD 0.999
Key: MTB: Mycobacterium Tuberculosis; +Ve: Positive; IPT: Isoniazid Preventive Therapy; RT: Retreatment;
OR: Odds Ratio; C.I: Confidence Interval
Further the findings revealed that there was no significant difference between TB false positivity and the
Mycobacterium tuberculosis detection levels (high, medium, low and very low) gender and age of the patients
(Table 1). In all age categories of the isoniazid preventive therapy arm there were 0(0%) false positive TB cases
in males and females respectively, the study further revealed that there were 3(4.5%) and 2(3%) false positive
TB cases in male and female patients from the non-isoniazid preventive therapy arm respectively, in the age
category (20-39) (Table 2). This indicates the age category 20-39 was more prone to false positive TB cases,
and males had higher rate of false positivity than females having 4.5% and 3% respectively.
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Table 2: Distribution of false positives tuberculosis Cases among study patients
True Xpert MTB/RIF
assay Positive results;
False Xpert MTB/RIF
assay positive results;
Total
IPT status Gender
MTB+ve /Growth MTB+ve /No Growth
IPT
patients
Male
Age
(Years)
60+ 1(10%) 0(0%) 1
40 – 59 2(20%) 0(0%) 2
20 – 39 3(30%) 0(0%) 3
Total 6(60%) 0(0%) 6
Female
Age
(Years)
60+ 1(10%) 0(0%) 1
40 – 59 1(10%) 0(0%) 1
20 – 39 1(10%) 0(0%) 1
< 20 1(10%) 0(0%) 1
Total 4(40%) 0(0%) 4
Total
Age
(Years)
60+ 2(20%) 0(0%) 2
40 – 59 3(30%) 0(0%) 3
20 – 39 4(40%) 0(0%) 4
< 20 1(10%) 0(0%) 1
Total 10(100%) 0(0%) 10
Non IPT
patients
Male
Age
(Years)
60+ 1(1.5%) 0(0%) 1
40 – 59 12(17.9%) 0(0%) 12
20 – 39 24(35.8%) 3(4.5%) 27
< 20 1(1.5%) 0(0%) 1
Total 38(56.7%) 3(4.5%) 41
Female
Age
(Years)
40 – 59 8(11.9%) 0(0%) 8
20 – 39 14(20.9%) 2(3%) 16
< 20 2(3%) 0(0%) 2
Total 24(35.8%) 2(3%) 26
Total
Age
(Years)
60+ 1(1.5%) 0(0%) 1
40 – 59 20(29.9%) 0(0%) 20
20 – 39 38(56.7%) 5(7.5%) 43
< 20 3(4.5%) 0(0%) 3
Total 62(92.5%) 5(7.5%) 67
Total 72 5 77
Key: IPT: Isoniazid Preventive Therapy; +Ve: Positive; MTB: Mycobacterium Tuberculosis
Discussion
Xpert MTB/RIF assay is a molecular technique
widely used currently all over the World for
diagnosis of active tuberculosis (TB) [1]. This
assay’s positivity for Mycobacterium tuberculosis
deoxyribonucleic acid (DNA) can remain detected
for years after treatment in the absence of viable
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organisms for culture [4]. Understanding these
Xpert false positive results is of paramount
importance given the large global burden of
symptomatic patients who present in health
institutions for investigation of active TB but have
previously been treated for active TB disease [4].
The current study revealed that 25% (P=0.001)
false positive TB cases (MTB detection on Xpert
MTB/RIF assay and negative result on BACTEC
MGIT 960 culture) among previously treated TB
patients, while there were 0(0%) false positive TB
cases among the new patients. These study findings
revealed significant association between TB false
positivity and retreatment TB patients, but there
was no significant association between TB false
positivity and the IPT status of the patients, MTB
detection levels, age and gender of the patients.
These findings concur with previous studies where
the rate of TB false positivity among retreatment
patients was South Africa 14% [14] 7 % [4], Egypt
2% [15], India 5.3% [16] and China 0.8% [17].
The current study findings confirm with other
previous studies that false positive TB cases are
significantly associated with previously treated TB
patients who had active TB disease. These findings
can be attributed to the fact that Xpert MTB/RIF
assay being a molecular technique cannot
differentiate between viable and non-viable
mycobacterial DNA, hence therefore the assay can
detect DNA from dead bacilli of previously treated
patients who had active TB disease [4]. Xpert
MTB/RIF assay results of this nature can lead to
baseless treatment of the patients, increase the
health care costs and delay in reaching the correct
diagnosis for the patients which can even lead to
death [4]. In this regard patients started on
medication based on wrong diagnosis due to false
positive TB results, may end up dying because of
wrong diagnosis, since the really problem remains
unknown and untreated. This calls for clinicians to
exercise caution when interpreting results from
previously treated TB patients [4].
Further the current study findings revealed that
there were 3(4.5%) false positive TB cases in male
cases, in the age category (20-39). These findings
concur with previous finding in Brazil where there
was 73.6% false TB positivity among male patients
[18]. These findings would be attributed to the fact
that males in this age category (20-39), exhibit
some characteristics which expose them to
recurrent episodes of TB disease, making them
vulnerable to false positivity because of harboring
deoxyribonucleic acid from the previous episodes
[18]. This characteristics include overcrowding
since most of these people are found in institutions
of learning, which are often crowded increasing
transmission of this air borne disease, peer pressure
which is common during this period leads them to
engage in irresponsible behavior like drug and
alcohol abuse coupled with the already depressed
immunity due to HIV makes the body vulnerable to
TB disease [18].
Conclusions
Xpert MTB/RIF assay positivity for Mycobacterial
DNA can remain detected for years after treatment
in the absence of viable organisms for culture [4].
Understanding such assay results is of paramount
importance given the huge global burden of
symptomatic patients who present for investigation
of active TB and have history of previous active
TB disease treatment. There was significant
association between previously treated TB patients
and TB false positivity. But TB false positivity was
not significantly associated with the MTB detection
levels, IPT status, gender or age of the patients.
Clinicians should wait confirmatory testing in
Xpert positive TB results for retreatment patients
before commencing them on treatment, they should
also take detailed history especially accurate
classification of the patient and this will lead to
proper patient management. GeneXpert should not
be used to follow up patients who are on treatment,
this is because the mycobacterial deoxyribonucleic
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Archives of Microbiology & Immunology Vol. 4 No. 2 – June 2020 64
acid will still be detected and the results will be
positive for MTB detection, therefore smear
microscopy still remains a major test in TB
treatment follow up. The findings of this study
have important policy implications which include
the gaps in TB management guidelines and the
need for revision and standardization to avoid
exposing patients to unwarranted treatment.
Recommendation
Studies should be conducted to monitor Xpert
MTB/RIF assay tuberculosis positive patients after
treatment completion to ascertain duration of
mycobacterial DNA survival, also clinicians should
be very careful when dealing with retreatment
patients to avoid baseless treatment which is not
only expensive but toxic.
Acknowledgement
The authors owe special gratitude to the staffs of
Central Reference Laboratory, Bahati
Comprehensive Care Centre staffs and all those
who directly or indirectly contributed to the success
of this study.
Competing interests
The authors declare that they have no competing
interests.
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Journal of Tuberculosis
Open Access | Research Article
Lucy Obonyo Nyang’au1*; Evans Amukoye2; Stanley Kangethe1; Jackson Onyuka1
1Mount Kenya University, Thika, Nairobi, Kenya
2Kenya Medical Research Institute, Nairobi, Kenya
ISSN: 2640-1193
MedDocs Publishers
2
Journal of Tuberculosis
Introduction
Mycobacterium Tuberculosis (MTB) is the causative agent of tuberculosis, which still remains a leading cause of mortality and morbidity globally [1]. Worldwide tuberculosis was top ten cause of death alongside Human Immunodeficiency Virus (HIV) in 2018 [2]. In 2018, 10.0 million people fell ill with tuberculosis; 1.2 million TB deaths were among HIV negative people while an additional 251,000 were among HIV positive people [2]. HIV co-infection has been associated with unusual presentations of TB such as smear negative and abnormal chest radiographs thus causing a diagnostic challenge, poor treatment outcome and subsequent increased mortality [3]. People living with HIV who have TB infection have a 5-10% annual risk of developing TB compared to 5-10% lifetime risk in HIV negative individuals [1]. Isoniazid Preventive Therapy (IPT) together with other interventions such as intensified case finding and infection control has been widely recommended to reduce the burden of TB in people living with HIV (PLHIV) [4].
Isoniazid preventive therapy has proven to be safe with minimal and less frequently reported side effects such as hepatotoxicity and gastrointestinal symptoms, studies have shown that IPT can lower TB incidence among people living with human immunodeficiency virus (PLHIV) by up to 70% if used with or without ART [4]. Taking isoniazid as a preventive measure is a cost effective and simple way that prevents TB if present to become inactive, the drug has been a standard for treatment of tuberculosis and preventive therapy due to its high potency, infrequent toxicity, low cost and non- bulk [4]. Treatment of Latent Tuberculosis Infection (LTBI) prevents its progression to active disease, both in HIV negative population and those infected with HIV [5]. Increasing uptake of isoniazid preventive therapy in HIV positive people prevents deaths and cases caused by tuberculosis [6]. Uptake of IPT has been relatively slow in most developing countries; it works synergistically with and independently of antiretroviral therapy (ART) to reduce tuberculosis morbidity, mortality and incidence among PLHIV [7,8].
Isoniazid preventive therapy involves provision of isoniazid tablets to those who meet the eligibility criteria [8]. The recommended dose is 10mg/Kg daily for children and up to 300mg/day for adults [8]. To end the global TB epidemic, it entails addressing the significant reservoir of TB infection, especially in PLHIV who have the highest risk of progression to TB disease [8]. Emphasis on TB prevention not only spares individuals the burden of TB associated morbidity and mortality but it also reduces the economic impact of the disease on the health system as a whole [9]. Isoniazid preventive therapy is protective towards progressing to active tuberculosis disease and retreatment tuberculosis patients are more prone to TB disease [1]. Although IPT is crucial and cost effective component of HIV care for adults and children and has been strongly recommended as an international standard of care for over a decade, it has remained highly underutilized [10]. Regular screening for TB disease among PLHIV is a standard of care and a critical component of HIV care and treatment because it can be effectively treated
Conclusion: Isoniazid preventive therapy is protective towards progressing to active tuberculosis disease and symptomatic previously treated Tuberculosis (TB) patients are more likely to have confirmed TB disease. There was no significant association between prevalence of tuberculosis, age and gender of the patients
especially when diagnosed early, hence therefore finding and treating people with TB disease and thus interrupting further transmission remains a top global healthy priority [8].
Materials and methods
Study design
This was a retrospective cohort study design conducted in Makadara sub-county, Nairobi, Kenya. Eligible HIV positive participants (with or without use of IPT) were recruited through cluster random sampling, only those who gave consent were enrolled in the study.
Inclusion and exclusion criteria
Patients who were HIV positive, on care, above 15 years, with signs and symptoms of tuberculosis, at one year post IPT, were included in the study upon consent. While patients with IPT and age records not clear, unable to consent, with other samples other than sputum were excluded from the study.
Sputum collection
Good quality spot and morning sputum samples were collected in 50ml sterile conical tubes; the samples were processed according to the standard operating procedures for GeneXpert MTB/RIF assay and culture using BACTEC MGIT 960 machine.
Laboratory methods
Identification of mycobacterium tuberculosis
Good quality sputa [11] and the reagent buffer were mixed according to the standard operating procedure and loaded into the Xpert MTB/RIF assay cartridge and test started on Xpert MTB/RIF assay machine platform[3]. The assay has internal quality controls which serve to verify that lysis of Mycobacterium tuberculosis has occurred, sample preparation is adequate and detect any inhibitor of polymerase chain reaction; this is accomplished by the sample processing control (SPC) [3]. Sample processing control must be positive when the result reads Mycobacterium tuberculosis not detected, while it can be negative or positive when the result is Mycobacterium tuberculosis detected. The system undertakes to measure fluorescence signal, rehydrating the beads and checking stability of the probe and dye. This is accomplished by the probe check control [3]. Upon completion of the test, results were either of the following; Mycobacterium tuberculosis not detected, Mycobacterium tuberculosis detected very Low, Low, Medium or High. In this case the rifampicin resistance can be either detected or not detected. The test results can also be in form of error or invalid, in this case the test must be repeated [3].
Culture of mycobacterium tuberculosis
Sputa were subjected to culture for the presence of Mycobacterium tuberculosis (MTB) on non-radiometric method Mycobacterium Growth Indicator Tube (MGIT) BACTEC 960. This was done according to manufacturer recommendations. Decontamination of the sputa was done using sodium hydroxide solution (40% w/v) combined with 2.9% sodium citrate solution and N-acetyl-L-cystein powder [12,17]. Sterile phosphate buffer was added and the organisms concentrated by centrifugation at 3,000 rpm for 15 minutes. The supernatant was decanted and the sediment suspended with phosphate buffer and inoculated in liquid MGIT media and incubated along with negative control (un-inoculated MGIT media) and positive control (H37Rv ATCC 27294) [12]. The inoculated MGIT tubes were incubated in the
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BACTEC MGIT 960 machine at 37 oC until the instrument flagged them positive. After a maximum of six weeks, the instrument flagged the tubes negative only if there was no growth at 37 oC [13]. Isolates from MGIT 960 were subjected to confirmative identification of MTB using BD MGIT TBc. Positive culture for MTB confirmed diagnosis of active disease [13,14].
Quality control
Un-inoculated Mycobacteria growth indicator tube (MGIT) (negative control), and H37Rv ATCC, 27294(positive control) were processed and included during the run. Purity of bacterial suspensions used was checked by culture on blood agar [15,16].
Statistical analysis
Data was entered and analyzed by SPSS version 20.0 statistical software. Pearson’s Chi-square test was applied to determine the differences in proportion for both groups in IPT status, type of patient and gender against the detection of MTB. Pearson’s Chi-square test was applied to determine the differences in proportion for both groups in IPT status, type of patients and demographics against TB. These results were presented by appropriate tabulations based on the determined variables, odds ratio (OR) with 95% confidence interval (CI) and the corresponding P- values. The threshold for statistical significance was set at P≤ 0.05.
Results
Prevalence of Tuberculosis (TB) in relation to Isoniazid Preventive Therapy (IPT) and patient treatment status
Of the 346 sputum samples subjected to Xpert MTB/RIF assay analysis, 10(6.5%) and 67(35.1%) had Mycobacterium tuberculosis(MTB) detected in IPT and non-IPT patients respectively, (OR 7.835[95% C.I =3.866-15.878]; P=0.0001) (Table 1), on the other hand new and retreatment patients recorded 57(18.2%) and 20(60.6%) detection of MTB respectively,(OR 0.145[95% C.I=0.068-0.308]; P=0.0001) (Table 1). This indicated a significant association between the IPT status of the patient and MTB detection; the non-IPT arm of patients was more prone to TB, than the IPT arm. On the other hand there was a significant association between MTB detection and patient treatment status; retreatment TB patients were more prone to TB disease than the new patients. Further findings revealed that detection of TB was high among the males of age category (20-39), 27(14.1%) from the Non- IPT arm, in the same age category females documented 16(8.4%) TB cases (Table 2). There was no significant association between prevalence of TB and the age or gender of the patients. Study findings also revealed that 145(93.5%) and 124(64.9%) samples were negative for MTB among IPT and Non-IPT patients while 256(81.8%) and 13(39.4%) samples were negative among new and retreatment patients.
Table 1: Mycobacterium tuberculosis results from Xpert MTB/RIF Assay among study patients
Variables
Total (N)
MTB Detected n (%)
MTB Not Detected n (%)
OR (95% CI)
P Value
IPT Status
IPT Patients
155
10 (6.5)
145 (93.5)
7.835 (3.866-15.878)
0.0001
Non-IPT Patients
191
67 (35.1)
124 (64.9)
Patient Treatment Status
New Patients
313
57 (18.2)
256 (81.8)
0.145 (0.068-0.308)
0.0001
RT Patients
33
20 (60.6)
13 (39.4)
Gender
Female
153
30 (19.6)
123 (80.4)
0.758 (0.452-1.271)
0.293
Male
193
47 (24.4)
146 (75.6)
Age(Years)
< 20
34
4 (11.8)
30 (88.2)
20 – 39
191
47 (24.6)
144 (75.4)
0.791 (0.62-2.333)
0.624
40 – 59
103
23 (22.3)
80 (77.7)
0.613 (0.423-3.899)
0.454
60 +
18
3 (16.7)
15 (83.3)
0.696 (0.332-0.78)
0.591
MTB: Mycobacterium Tuberculosis; RT: Retreatment; IPT: Isoniazid Preventive Therapy; OR: Odds Ratio;
CI: Confidence Interval
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MTB DETECTION
Total
IPT Status
Gender
MTB DETECTED
MTB NOT DETECTED
IPT Patients
Male
Age (Years)
60+
1(0.6%)
4 (2.6%)
5
40 – 59
2(1.3%)
19 (12.3%)
21
20 – 39
3(1.9%)
43 (27.7%)
46
< 20
0(0%)
8 (5.2%)
8
Sub-Total
6(3.9%)
74 (47.7%)
80
Female
Age (Years)
60+
1(0.6%)
4 (2.6%)
5
40 – 59
1(0.6%)
22 (14.2%)
23
20 – 39
1(0.6%)
33 (21.3%)
34
< 20
1(0.6%)
12 (7.7%)
13
Sub-Total
4(2.6%)
71 (45.8%)
75
Total
Age (Years)
60+
2(1.3%)
8 (5.2%)
10
40 – 59
3(1.9%)
41 (26.5%)
44
20 – 39
4(2.6%)
76(49%)
80
< 20
1(0.6%)
20(12.9%)
21
Total
10(6.5%)
145 (93.5%)
155
Non IPT patients
Male
Age (Years)
60+
1(0.5%)
3 (1.6%)
4
40 – 59
12(6.3%)
31 (16.2%)
43
20 – 39
27(14.1%)
35 (18.3%)
62
< 20
1(0.5%)
3 (1.6%)
4
Sub-Total
41(21.5%)
72 (37.7%)
113
Female
Age (Years)
60+
0(0%)
4 (2.1%)
4
40 – 59
8(4.2%)
8(4.2%)
16
20 – 39
16(8.4%)
33 (17.3%)
49
< 20
2(1%)
7 (3.7%)
9
Sub-Total
26(13.6%)
52 (27.2%)
78
Total
Age (Years)
60+
1(0.5%)
7( 3.7%)
8
40 – 59
20(10.5%)
39 (20.4%)
59
20 – 39
43(22.5%)
68 (35.6%)
111
< 20
3(1.6%)
10 5.2%)
13
Total
67(35.1%)
124 (64.9%)
191
Total
77
269
346
Table 2: Xpert MTB/RIF Assay Results in relation to IPT status, Gender and Age
IPT: Isoniazid Preventive Therapy; RIF: Rifampicin; MTB: Mycobacterium Tuberculosis
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Discussion
The current study findings indicate that there is significant association between the isoniazid preventive therapy(IPT) status of the patient and likelihood of developing active Tuberculosis disease (TB); the non-IPT arm of patients had a significantly high rate of tuberculosis(TB) cases (35.1%) compared to the IPT arm (6.5%), these study findings confirm with other previous findings that IPT protects against progressing to active TB in the risk populations, among them being people living with HIV. The current study findings were relatively higher than those observed in previous studies in South Africa (15.9%) [18], Zimbabwe (3.0%) [8], Indonesia (Hasan Sadikin hospital) (4.3%) [9] Ethiopia 16.32% [4], (27.8%) [19] (13.4%) [5], (8.8%) [1]. On the other hand the findings were comparable with results from a previous study conducted in Egypt (Al- Hussein university) (36.7 %) [20], but the findings were relatively low than previous study findings in Pakistan 49.46% [21], (98.7) [22].
The difference in the findings can be attributed to low uptake of isoniazid preventive therapy. The study findings further indicated significant difference in the number of TB cases among patient treatment status; retreatment patients had (60.6%) while new patients had (18.2%), there was a significantly high rate of TB cases among the retreatment cases. The current findings confirm with other previous studies that retreatment patients are significantly associated to TB disease. The current study findings are slightly high than those documented in Ethiopia (53.8%) [23], on the other hand these findings are markedly high than previous findings in a study conducted in Benin (6%) [24], this difference may be attributed to lack of patient compliance to TB drugs, treatment interruptions due to either (imprisonment, defaulting) and weak active case finding strategies which allow re-infection of the already cured people, HIV prevalence [23]. Further findings of the current study indicated that a high percentage of male patients of age category 20-39 years had TB (14.1%), further the findings were markedly lower than those recorded in Thailand (43.7%) [25]. On the other hand the study findings were comparable with those recorded in Ethiopia (14.2%) [19].
These finding would be attributed to the fact that in this age category (20-39), people are normally in institutions of learning, which are often crowded increasing transmission of this air borne disease. Also because of peer pressure which is common during this period, engaging in irresponsible behavior like drug abuse coupled with the already depressed immunity due to HIV makes the body vulnerable to TB disease [10]. The health service seeking behavior can also be attributed to the high TB cases in males of this age category (they tend to seek health services only when the condition is serious) and the mobility they exhibit; in African culture males are supposed to fend for the family thus they have to move seeking for menial jobs in the process they are exposed to conditions which predispose them to get TB [26]. 20-39 years is the productive age of an individual, TB disease being chronic will compromise productivity of this population in the sectors they offer their services and thus decrease a country’s gross domestic product which eventually will lead to an unstable economy [10]. In institutions of learning students will drop out because of either stigma from friends and the lecturers or the challenges which come along with the long TB treatment duration, this might lead to a country lacking work force because the productive population will lack the necessary skills because of lack of education, worse more because of stigma treatment defaulting is high and this might lead to increased mortality and morbidity [10]. One important limitation of this study is that it involved participants who consented to be part of the study; the non-participants would not be profiled.
Conclusions
IPT protects against development of active Tuberculosis (TB) disease in eligible patients, who harbor latent TB. Retreatment patients were significantly associated with TB because of either lack of adherence to medications during previous TB treatments or interruptions during treatment which lead to lack of treatment completion. Of the patients infected with the disease a high percentage (14.1%) was male of age 20-39 years and those patients from the non-IPT arm.
Recommendation
Call for intensified efforts towards increasing the uptake of isoniazid preventive therapy in patients who are eligible to curb progressing of latent tuberculosis to active disease, especially in vulnerable population like people living with HIV. Active case finding on index cases should be made routine to avoid re-infections for those already cured. In this case there should be a budget for purposes of tracing the contact cases in their homes. Through the ministry of health friendly health education should be put in place, mostly in social gatherings to inform the population on what is TB, how it is spread, prevented, diagnosed, treated and importance of seeking medical attention earlier to avoid complications and further spread of the disease. This should be done in such a way that the target should be males 20-39 years of age.
Acknowledgement
The authors sincerely thank Central Reference Laboratory, Bahati Comprehensive Care Centre staffs and all those who directly or indirectly contributed to the success of this study.
References
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4. Geremew D, Endalamaw A, Negash M, Eshetie S, Tessema B. The protective effect of isoniazid preventive therapy on tuberculosis incidence among HIV positive patients receiving ART in Ethiopian settings: a meta-analysis. BMC Infectious Diseases. 2019; 19:405.
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International Journal of Sciences:
Basic and Applied Research
(IJSBAR)
ISSN 2307-4531
(Print & Online)
http://gssrr.org/index.php?journal=JournalOfBasicAndApplied
---------------------------------------------------------------------------------------------------------------------------
Determining First Line Anti-Tuberculosis Drug Resistance among New and Re-treatment Tuberculosis/ Human Immunodeficiency Virus Infected Patients, Nairobi Kenya Lucy Obonyo Nyang’aua*, Dr. Evans Amukoyeb, Prof. Zipporah Ng’ang’ac aInstitute of Tropical Medicine and Infectious Diseases (ITROMID), Jomo Kenyatta University of Agriculture and Technology (JKUAT), Box 4899-00200 Nairobi, Kenya. bCentre for Respiratory Diseases Research, Nairobi, Kenya (CRDR), Kenya Medical Research Institute, Kenya Medical Research Institute cJomo Kenyatta University of Agriculture and Technology (JKUAT), Nairobi, Kenya
alucynyangau@yahoo.com
bamukoye@gmail.com
czipnganga@gmail.com Abstract Drug resistant tuberculosis (T.B) is a state when Mycobacterium tuberculosis (MTB) organisms are resistant to antimicrobial agents at the levels attainable in blood and tissue. Scarce data exists on the prevalence of resistance to first line anti-tuberculosis drugs in populations with high rates of tuberculosis and human immunodeficiency virus (H.I.V). Strains of MTB complex from MGIT were subjected to drug susceptibility testing for isoniazid (INH), Rifampicin (R), Streptomycin(S), and Ethambutol (E) using the proportional method on (MGIT). A total of 145 TB patients were enrolled for study. Of the 138 patients who had valid results for analysis, 79(57.2%) were male and 59(42.8%) were female. ------------------------------------------------------------------------ * Corresponding author. E-mail address: lucynyangau@yahoo.com.
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Most of the patients (20.3%) were aged between 35-39 years with the lowest proportion (3.6%) being in the younger category <20 years. Among the pulmonary tuberculosis patients 34% were new cases while 66% were retreatment cases. A total of 43(31.2%) strains showed resistance to at least one drug tested, while 112(81.2%) were susceptible. The isolates showed different resistance patterns with mono-resistance in 15(11%) isolates, total multi- drug resistance (MDR) in 6(4.3%) isolates with new and retreatment cases being 0(0.0%) and 6(6.6%) respectively. Mono-resistance was recorded in all four drugs tested. The isolates were resistant to the antibiotics as follows; 16(17.6% and 0(0.0%) were resistant to INH; 9(9.9%) and 0(0.0%) were resistant to R; 10(11.0%) and INH (2.1%) were resistant to E; 7(7.7%) and 0(0.0%) were resistant to S; 6(6.6%) and 0(0.0%) were multi drug resistant among retreatment and new cases respectively. Our study concluded that there were high levels of drug resistance among those previously treated for TB.
Keywords: Multi drug resistant tuberculosis; New and Retreatment; HIV; Resistance
1. Introduction
Tuberculosis is a serious public health problem, a third of the world population that is two billion people are infected with TB, 9 million develop TB disease and close to 2 million die annually, 650,000 develop MDR-TB while 8% are co-infected with HIV [14]. 95% of these TB cases occur in developing countries, where 1 in 14 new cases occur in individuals who are infected with HIV and 85 % of these cases occur in Africa [10]. According to the WHO global report of 2008, about 9.2 million new cases and 1.7 million deaths from TB occurred in 2006 and of these around 709,000 (7.7%) new cases and 200,000 deaths were estimated to have occurred in HIV positive individuals [12]. Highest rates of TB are reported in the countries of Eastern Europe, where weakened economies and public health efforts are the main causes of its resurgence, and where internationally recommended control strategies need further expansion and strengthening. In Western Europe, there are pockets of increasing incidence, particularly in major cities with socially marginalized immigrants from high burden TB countries [13-4]. Studies on drug resistance in various countries in the 1960’s showed that developing countries had a much higher incidence of drug resistance than developed countries3. Resistance to (INH) and (S) was more than resistance to (R) and (E) and the rate of primary drug resistance to (INH) as a single agent ranged from 0% to 16.9% among HIV infected individuals [6]. (INH) forms the core of anti-tuberculosis drugs, and its use in TB preventive therapy has been known to reduce incidence in high risk individuals for more than 40 years [7]. Despite the confirmed efficacy of preventive therapy, concerns about drug resistance, have limited its uptake [7].
HIV infection by impairing the cell mediated immunity is the most potent known risk factor for the reactivation of latent TB infection and rapid progression to active disease [8]. Overall an estimated 8% of new cases are attributable to HIV co- infection [14]. An estimated 13% of the 1.5 million TB deaths in 2010 were attributed to HIV infection, but in the African region this proportion has been much higher because of the high HIV prevalence [2]. The risk of death in co-infection is twice that of HIV infected individuals without TB, even when CD4 cell count and ART therapy are taken into account [2-16]. Some groups of people are at higher risk for TB disease because they are more likely to be exposed to or infected. Risk of infection is; poor housing and crowding, large pool of untreated persons. Risk of developing disease after infection is increased by low 427
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immunity (extreme of ages, HIV, diabetes). This includes close contacts of people with infectious TB disease, people in areas where TB is common, elderly, drug users, and people with certain medical conditions especially HIV infections. For people with HIV and TB, the risk of developing TB disease is about 10% each year, in contrast, people infected only with TB; the risk of developing TB disease is 10% over a lifetime [13]. Control of drug resistance involves; identifying and treating drug resistant cases, treating all cases, treating latent TB, improving cure rate, active case finding, and reducing development of secondary drug resistance by improving adherence [5].
Introduction of the first anti-tuberculosis drugs, (INH), (S), (PAS), was slowly followed by resistance, which was observed in clinical isolates of MTB [3]. Over 60% of new cases of pulmonary TB in most developing countries are now co-infected with HIV [9-1]. The WHO approach (identifying of TB bacilli microscopically) to TB diagnosis is failing in a number of HIV infected patients, as smear negative TB has been linked to poor treatment outcomes, including death [9]. According to the national MDR-TB surveillance data 2011, approximately 150 HIV positive individuals were diagnosed with MDR-TB and two cases confirmed with extremely drug resistance tuberculosis (XDR-TB) in Kenya [18]. These figures have been on the rise because of inadequate and insensitive diagnostic methods, leading to increased mortality and morbidity among those infected [5]. This study seeks to determine first line anti-TB drug resistance among HIV infected patients attending Maryland comprehensive care centre.
This study was undertaken to determine M. tuberculosis resistance patterns against first-line drugs used for treatment in patients diagnosed with pulmonary TB and living with HIV.
2. Materials and Methods
2.1 Setting
The study was conducted in Mathare 4A, Nairobi the capital city of Kenya. The population of Mathare is nearly 150,000 and is steadily growing due to rural /urban migration. This poses a lot of challenges. A significant proportion of the residents of Mathare belong to low economic social class, with high population densities. Mathare valley is approximately 6km to the north east of Nairobi’s central business district. It is bordered by Thika road to the north and Juja road to the south. The study was cross sectional, eligible patients (new and retreatment) randomly sampled during the intake period, who gave consent were enrolled for the study. The study consisted of 79(57.2%) male and 59(42.8%) female patients. The intake period was between April and November 2013.
2.2 Specimen Collection and Transport
One early morning sputum and a spot sample were collected on screw capped bottles. Genexpert was done on the first sample to confirm T.B diagnosis of suspected patients. Second samples from the MTB positive patients were then transported weekly by smith-line courier service, to central reference laboratory (CRL) for culture and drug susceptibility testing (DST). The CRL is located within the centre for respiratory diseases research, Kenya Medical Research Institute (CRDR-KEMRI) at Kenyatta National Hospital.
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2.3 Culture of M. Tuberculosis and Drug Susceptibility Testing
Sputum culture and DST for M. tuberculosis was conducted in the central reference laboratory. Primary culture of M. tuberculosis was performed using non radiometric method Mycobacterium growth indicator tube (MGIT) 960. The sputa were decontaminated with NAOH solution (40%w/v) combined with 2.9% sodium citrate solution and N-acetyl -L-cystein (NALC) powder. Sterile phosphate buffer was added and the organisms concentrated by centrifugation at 3,000rpm for 15 minutes. The supernatant was decanted and the sediment suspended with phosphate buffer and inoculated in liquid MGIT media and incubated along with a growth control and an external control H37Rv at 370C in BACTEC 960 systems (BD Diagnostic Systems, Sparks, MD, USA). The MGIT tubes were incubated until the instrument flagged them positive. After a maximum of six weeks, the instrument flagged the tubes negative if there was no growth at 370C. A positive culture of M. tuberculosis confirmed the diagnosis of active disease.
2.4 Sensitivity Testing of M. tuberculosis
All culture positive tubes were tested for contamination before sensitivity tests using the standard method used in Kenya for drug susceptibility BACTEC MGIT 960 liquid culture system (Becton Dickson Company Sparks, MD, USA. A total of four first line drugs collectively referred to as SIRE (Streptomycin (S)-1.00ug/ml; Isoniazid (I)-0.10ug/ml; Rifampicin (R)-1.00ug/ml and Ethambutol (E)-5.00ug/ml were tested for sensitivity. A control tube was matched with all the isolates tested. An external control of H37Rv was also set in all culturing and sensitivity testing processes. All readings were performed inside the machine and results were printed as susceptible, resistant or indeterminate.
3. Ethical Approval
The research proposal was approved and ethically cleared by the national ethical review committee (ERC) and Scientific Steering Committee (SSC) at the Kenya Medical Research Institute (KEMRI). Each patient who consented to enroll was required to complete an informed consent form.
4. Results
A total of 145 patients were enrolled for study in the Maryland comprehensive care centre (Figure 3). Of the 138 patients for whom data was available, 47(34.1%) were new and 91(65.9%) previously treated for TB. A total of 138 sensitivity tests were performed from pulmonary tuberculosis patients of whom 79(57.2%) were male and 59(42.8%) were female (Figure 2). Most of the patients (20.3%) were aged between 35-39 years with the lowest proportion (3.6%) being in the younger category <20 years (Figure 1).
A total of 26(18.8%) strains showed resistance to at least one drug tested, while 112(81.2%) were susceptible. The isolates showed different resistance patterns with mono-resistance in 15(11%) isolates, total multi- drug resistance in 6(4.3%) isolates with new and retreatment cases being 0(0.0%) and 6(6.6%) respectively. Mono-resistance was recorded in all four drugs tested. 429
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The isolates were resistant to the antibiotics as follows; 16(17.6% and 0(0.0%) were resistant to INH; 9(9.9%) and 0(0.0%) were resistant to R; 10(11.0%) and 1NH (2.1%) were resistant to E; 7(7.7%) and 0(0.0%) were resistant to S; 6(6.6%) and 0(0.0%) were multi drug resistant among retreatment and new cases respectively. Seventeen (12.3%) males and 9(6.5%) female patients showed resistance to at least one drug tested (Table 1). The proportion of patients that was sensitive to all drugs among the retreatment cases (72.5%) was significantly low compared to the new cases (97.9%), (OR=0.06 [95% CI=0.01 – 0.44]; p<0.001). Implying that, a retreatment case was 94.0% less likely to be sensitive to all drugs compared to a new case. Resistance to Isoniazid and Rifampicin was significantly associated with TB retreatment (P=0.001, and P=0.028 respectively) (Table 1).
Table 1: Patterns of resistance to first line anti-tuberculosis drugs in relation to treatment status
Antibiotic
Total (n=138)
RT (n=91)
New (n=47)
OR
95% CI
p value
N
%
N
%
N
%
Lower
Upper
Sensitivity to all
112
81.2%
66
72.5%
46
97.9%
0.06
0.01
0.44
<0.001
Any resistance pattern
Isoniazid (H)
16
11.6%
16
17.6%
0
0.0%
UD
UD
UD
0.001
Rifampicin (R)
9
6.5%
9
9.9%
0
0.0%
UD
UD
UD
0.028
Ethambutol (E)
11
8.0%
10
11.0%
1
2.1%
5.68
0.70
45.79
0.098
Streptomycin (S)
7
5.1%
7
7.7%
0
0.0%
UD
UD
UD
0.095
Monoresistance TB
Isoniazid (H)
6
4.3%
6
6.6%
0
0.0%
UD
UD
UD
0.095
Rifampicin (R)
2
1.4%
2
2.2%
0
0.0%
UD
UD
UD
0.548
Ethambutol (E)
4
2.9%
3
3.3%
1
2.1%
1.57
0.16
15.50
1.000
Streptomycin (S)
3
2.2%
3
3.3%
0
0.0%
UD
UD
UD
0.551
Multi drug resistance TB (MDR TB)
H+R
2
1.4%
2
2.2%
0
0.0%
UD
UD
UD
0.548
H+R+E
2
1.4%
2
2.2%
0
0.0%
UD
UD
UD
0.548
H+R+S
1
0.7%
1
1.1%
0
0.0%
UD
UD
UD
1.000
H+R+E+S
1
0.7%
1
1.1%
0
0.0%
UD
UD
UD
1.000
Total MDR TB
6
4.3%
6
6.6%
0
0.0%
UD
UD
UD
0.095
Other resistant Patterns
H+E
2
1.4%
2
2.2%
0
0.0%
UD
UD
UD
0.548
H+S
1
0.7%
1
1.1%
0
0.0%
UD
UD
UD
1.000
H+E+S
1
0.7%
1
1.1%
0
0.0%
UD
UD
UD
1.000
R+E
1
0.7%
1
1.1%
0
0.0%
UD
UD
UD
1.000
E+S
0
0.0%
0
0.0%
0
0.0%
-
-
-
-
R+S
0
0.0%
0
0.0%
0
0.0%
-
-
-
-
R+E+S
0
0.0%
0
0.0%
0
0.0%
-
-
-
-
Table 1; presents patterns of resistance to first line anti-tuberculosis drugs in relation to treatment status.
430
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Age distribution among the patients was normally distributed (Figure 1) with a mean age of 35.0 (+10.0 SD), and a median age of 35.0, ranging between 16 and 61 years. Figure 1 presents the distribution of study patients by age categories. Most of the Clients (20.3%) were aged 35-39 years with the lowest proportion (3.6%) being in the younger category (Age<20 years).
Figure 1: Age distribution among the study patients
Figure 2, presents distribution of gender among the study patients with tuberculosis. 79 (57.2%) and 59 (42.8%) of the patients were male and female respectively. The majority of the cases were male.
Figure 2: Gender distribution among the study patients
431
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Figure 3, presents a flow diagram on patient recruitment. Seven patients were removed from the study due to various reasons namely death, withdrawal, and culture contamination.
Figure 3: Flow diagram on Patient recruitment
5. Discussion
T.B drug resistance is a serious public health, and worldwide problem and a major challenge in T.B care and control. H.I.V infection is the major risk factor for the reactivation of latent TB infection and rapid progression to active disease [15].
5.1 Resistance Patterns
The overall resistance to all the drugs tested 18.8% was significantly lower to that reported in earlier studies in Kenya, where 30.1% isolates were resistant to at least one drug [19]. The results of this study differ with that conducted in Central Asia where resistance was 30.5% [24]. Resistance rates in the present study were slightly higher than rates observed in studies in Tanzania where only 14 out of 280 (5.83%) isolates were resistant to at least one drug [20], while in South Africa and Korea total resistance to the drugs tested was 7.4% [21] and 18.7% [22] respectively.
The results of the present study show a high rate of resistance among retreatment cases, with decreased resistance among new cases (46.5% and 2.1% respectively, while MDR-TB prevalence was 0.0% and 6.6% among new and previously treated patients respectively). This is comparable with studies conducted in Uganda where prevalence of resistance to any of the first-line anti-TB drugs was 8.3% and 25.9%; with an MDR-TB prevalence of 1.4% and 12.1% among new and retreatment cases respectively [31], in the Republic of Tanzania any resistance was 8.3% and 20% among new and retreatment cases respectively [23]. While in Kenya any
Total patients Recruited: 145
Available patients with results: 138
Died: 1
Withdrew at will: 2
Culture became contaminated: 4
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resistance was 21% and 45% among new and retreatment cases respectively [34]. All these were one-time studies performed in single facilities in the respective countries, similarly the present investigation was a one-time study performed in a single facility.
Resistance to (INH) in this study was 11.6% which was slightly lower than results obtained in earlier studies in Ethiopia, where one isolate was resistant to (INH) [25], with Bangladesh at 5.4% 26 and Sri-lanka at 12.2% [28]. According to WHO (INH) resistance rates higher than 10% can predict the development of MDR-TB. This high resistance may be caused by both its wide use in the treatment of TB as a first-line drug or poor compliance by patients. In this study resistance to (RIF) was 6.5% which is higher than that observed in earlier studies in Kenya, where resistance was 1.3 % [19]. This rate is also higher than reports from studies in Bangladesh where resistance was 0.5% [28] and Ethiopia where resistance to (RIF) ranged from 0% to 1.8% [20-22]. Rif has several adverse effects such as nausea, vomiting, rashes, hepatitis, GIT upset, flulike symptoms, fever, and jaundice, which could result in patients non adherence and hence may lead to the selection of resistant strains.
Resistance to (S) in this study was 5.1% which was comparable to the resistance of 5.2% [19] reported in another study in Kenya but lower than that reported in Ethiopia 26% [25] and Sri-Lanka 9.9% [27]. Resistance to (E) in this study was 8.0% which was higher than rates in Ethiopia 2.7% [25]. It was however; lower than studies conducted in Sri-lanka where 14.5% resistance was reported [27]. Ethambutol enhances the effect of many drugs including beta lactams to different Mycobacteria species and can be used to develop a regimen for MDR-TB [29]. In this study a high number of patients with TB showed IHN resistance but significantly susceptible to Rifampicin. It is therefore possible for these patients to recover fully if WHO guidelines for re-treatment are followed under strict supervision to prevent them from developing MDR-TB. However, the high rate of INH resistance is significant since it is a first line drug which is used throughout the course of treatment. This indicates a high probability for developing MDR-TB in the future since it has been observed that MDR often develops from initial INH mono-resistant strains. INH is also the drug of choice for chemoprophylaxis of TB and is used in developed countries for treating latent TB. The high level of INH resistance among the study population also is an indicator that this drug will be completely useless for both these purposes in Kenya.
In the present study MDR-TB prevalence was 0(0.0%) and 6(6.6%) among new and previously treated patients respectively. The result of this study compares with that of study conducted in Uganda where MDR-TB prevalence was 1.4% and 12.1% among new and previously treated patients respectively [32-18]. Higher levels of MDR-TB prevalence among retreatment’s cases raises concerns about the quality of directly observed therapy and adherence to treatment. The existence of very low primary resistance to INH, R, E, and S implies no ongoing transmission of drug resistant strains in the community. This would imply strengthened infection control measures which should therefore be further strengthened through dissemination of TB infection control guidelines by the National Leprosy and Tuberculosis Program (NLTP). Whereby priority should be accorded to TB infection control training for health care workers, in the TB diagnostic and treatment centre’s especially those which offer comprehensive TB/HIV care.
The scale up of Xpert MTB/RIF screening in Kenya will allow for a more expedient diagnosis of rifampicin resistant TB and may improve TB outcomes by shortening diagnostic delays and ineffective initial therapy [30-
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33]. The results of this study indicate the need for strict enforcement of the DOTS method and better epidemiological surveillance of Tb cases.
5.2 Comparison of Pulmonary TB and Resistance on basis of gender
There was a significantly high number of male diagnosed with TB than female (57.2% and 42.8% respectively). This compares with earlier studies in Kenya where more males were associated with pulmonary TB than female (60.4% and 39.5% respectively; p<0.05. Globally a 70% predominance of males over female patients was reported [20-16]. The world health organization reported that 67.2% of the global male population was diagnosed with TB as compared to the female population [35-15]. The great number of males compared to females could be attributed to behavioral factors such as smoking, which is a predisposing factor to TB with more males being smokers than females [36-2]. Alcohol consumption, malnutrition, and the delay to seek treatment, especially by men [36-37] are other factors that have been associated with higher numbers of males than females with TB, with 57% of the patients in the current study being male. In the present study more males were associated with drug resistance than females (17(12.3%) and 9(6.5%) respectively. This is consistent with earlier studies in Kenya, where more males were associated with drug resistance than females 52(18.2%) and 34(11.9%).
6. Study Limitation
This study was conducted over a short period of seven months and it was limited to Mathare, similar studies should be conducted in other regions.
7. Conclusion
Presence of MDR-TB should be awake up call to continue monitoring its course in addition to promoting the improvement and expansion of control activities. There is need for patients to access rapid diagnosis and rapid drug susceptibility tests and treatment with more effective drugs and also regimens shorter than the current two year period for MDR-TB. Introduction of rapid molecular diagnostic tests like Xpert MTB/RIF; makes diagnosis of MTB and identification of rifampicin resistance quicker, this makes patient management easier. DST of the other first line TB drugs E, S, H should be made available and accessible. There is a high prevalence of drug resistance among retreatment TB cases compared to new cases. This raises concerns about the quality of directly observed therapy and adherence to treatment. The fact that these patients are previously treated for TB could be a possible risk factor for the development of resistance due to incomplete and irregular treatment of tuberculosis in them.
Acknowledgement
We thank the Maryland comprehensive care centre, particularly the hospital laboratory staff for their support throughout the study period. We specially thank the KEMRI CRDR, SSC and ERC committees for approving the study. We also in a special way thank the patients for accepting to participate in the study and also for their patience throughout the study. 434
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