Exeley Inc.

Hult, A.K.

Olsson, M.L.

May the FORS be with you: a system sequel

Grąźlewska, Weronika

Ferra, Bartłomiej

Holec-Gąsior, Lucyna

PRZYDATNOŚĆ DIAGNOSTYCZNA REAKCJI PCR W ROZPOZNAWANIU BORELIOZY

Sheppard, C.A.

Bolen, N.L.

Meny, G.

Kalvelage, M.

Ochoa-Garay, G.

Concordance of two polymerase chain reaction–based blood group genotyping platforms for patients with sickle cell disease

ANTONENKO, Nadiia

RUMILETS, Tetiana

COVID-19 AND LIBRARY PUBLIC SPACE TRANSFORMATIONS

Fitzgerald, David

 
Aviation in general has been in turmoil this year with the outbreak of COVID-19 and the resultant travel 
restrictions.   Many airlines have cut back both in operational staff and airline medical departments are under 
pressure, albeit have taken on different roles and responsibilities.  Many operators have had to make 
significant adjustments to operations, as is reflected by the paper by Ganesh et al, noting the lessons learnt 
in RFDS operations during the outbreak. 
 
It is pleasing to see that there is still a healthy interest in aviation, and that interest continues in the 
aeromedical aspect of aviation.  In this year’s volume, we have a contributions from Masters in Aviation 
Medicine students who have undertaken literature reviews as part of their studies. 
 
A number of other papers are still under peer review and will be published early next year.  It has been a 
busy year for many of us, including those of us editors, peer reviewers and typesetters, and as editor in 
chief, I thank all for their contributions to the journal this year. 
 
We have now an editorial board in place to further strengthen the journal’s direction moving forward and 
thanks also to those who have agreed to serve on the board to support me in developing JASAM into a 
quality, well regarded source of aeromedical literature. 
 
As always, the journal invites submissions from those aeromedical examiners working in the field in terms of 
case reports that can be shared collegially with colleagues from around the world. 
 
Here’s hoping that 2021 brings some stability and re-growth to the aviation sector! 
 
David Fitzgerald 
Editor-in-Chief 
 


EDITORIAL

Prygiel, Marta

Mosiej, Ewa

Zasada, Aleksandra Anna

Polish

CAUSES OF PERTUSSIS INCIDENCE INCREASE

ALI, SAJID

KHAN, MUHAMMAD TAHIR

KHAN, ANWAR SHEED

MOHAMMAD, NOOR

KHAN, MUHAMMAD MUMTAZ

AHMAD, SAJJAD

NOOR, SADIQ

JABBAR, ABDUL

DAIRE, CANTILLON

HASSAN, FARIHA

Polish Journal of Microbiology
2020, Vol. 69, No 2, 133–137
https://doi.org/10.33073/pjm-2020-005
ORIGINAL PAPER
* Corresponding author: S. Ali, Quaid-i-Azam University, Islamabad, Pakistan; e-mail: sajidjan@live.com
© 2020 Sajid Ali et al.
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 License (https://creativecommons.
org/licenses/by-nc-nd/4.0/).
Introduction
Tuberculosis (TB) is a pre-historic disease caused 
by Mycobacterium tuberculosis (MTB) (Daniel 2006). 
Although there are more than 150 species of Mycobacte-
rium sp., MTB is still the most dominant and prevailing 
member of this genus all over the world, accounting for 
10 million deaths in 2019 (WHO 2019).
The World Health Organization (WHO) declared 
TB as a global emergency in 1993 (Grange and Zumla 
2002). Despite significant medical and social interven-
tions, TB consistently affects vulnerable populations 
across the world and remains a leading global public 
health problem. Treatment of drug-susceptible TB takes 
six months while treatment of rifampicin-resistant 
TB (RR-TB) and multidrug-resistant TB (MDR-TB) 
requires a long therapy for up to two years (WHO 2019).
Globally, an 85% successful treatment rate has 
been reported for drug-susceptible MTB. The emer-
gence of drug resistance, however, still poses a threat 
to global efforts. The WHO estimated 10.4 million 
new TB cases consist of 490 000 multidrug-resistant 
TB and 110 000 rifampicin-resistant TB. Five coun-
tries such as India, China, Indonesia, Philippines, and 
Pakistan are accounting for 56% of TB burden. Despite 
the development of rapid molecular tools, only 37% of 
MDR-TB were reported globally which shows labora-
tory gaps. China, India, and Russia reported 47% of 
the total global MDR/RR-TB cases. Pakistan is a high 
TB endemic country, standing at 5th position in the list 
Prevalence of Multi-Drug Resistant Mycobacterium Tuberculosis
in Khyber Pakhtunkhwa – A High Tuberculosis Endemic Area of Pakistan
SAJID ALI1*, MUHAMMAD TAHIR KHAN2, ANWAR SHEED KHAN3  , NOOR MOHAMMAD3,
MUHAMMAD MUMTAZ KHAN4, SAJJAD AHMAD3, SADIQ NOOR4, ABDUL JABBAR4,
CANTILLON DAIRE5 and FARIHA HASSAN1
1 Quaid-i-Azam University, Islamabad, Pakistan
2 Capital University of Science and Technology, Islamabad, Pakistan
3 Provincial TB Control Program, Khyber Pakhtunkhwa, Pakistan
4 University of Haripur, Khyber Pakhtunkhwa, Pakistan
5 Brighton and Sussex Medical School, University of Sussex, Brighton, United Kingdom
Submitted 24 October 2019, revised 20 December 2019, accepted 8 January 2020
A b s t r a c t
Anti-tuberculosis therapy involves the combination of drugs to hamper the growth of Mycobacterium tuberculosis (MTB). The emergence 
of multidrug-resistant tuberculosis (MDR-TB) is a global concern. Pakistan has been ranked 5th position in terms of a high burden of 
MDR-TB in the world. The aim of the current study was to investigate the prevalence of drug resistance in MTB in Khyber Pakhtunkhwa. 
Random samples were collected from 25 districts using the simple random sampling formula. All samples were processed in a biosafety 
level 3 laboratory for culture and drug susceptibility testing. Among 5759 presumptive tuberculosis (TB) cases, 1969 (34%) were posi-
tive. The proportion of TB was higher in females (39%) than males (29%), thus it represents a significant association between gender and 
tuberculosis (p < 0.05). People ages between 25 to 34 years were more likely to be infected with MTB (40%). Drug-resistant profile showed 
97 (4.9%) patients were infected with MDR-TB. Streptomycin resistance was the highest and was observed in 173 (9%) isolates followed 
by isoniazid in 119 (6%) isolates. The lowest resistance was observed to pyrazinamide (3%). The prevalence of MDR-TB (10.4%) among 
patients that previously received anti-tuberculosis treatment is seemingly high. A large-scale drug resistance survey is required to evaluate 
the drug resistance for better management of tuberculosis.
K e y  w o r d s: tuberculosis, MDR MDR-TB, multi drug-resistant TB
Ali S. et al. 2134
of 30 high burden countries (HBC) with an estimated 
518 000 TB cases including 15 000 MDR-TB. The esti-
mated proportion of MDR-TB is 4.2% in new patients 
and 16% in the previously treated patients (WHO 
2019). According to the drug resistance survey con-
ducted in 2012, the prevalence of MDR-TB was 3.7% in 
newly diagnosed TB cases and 18.1% among previously 
treated TB cases (Tahseen et al. 2016). KPK is one of the 
four provinces of Pakistan that contributes a propor-
tion of 11.9% in the total national population with an 
estimated 270 TB cases per 100 000 population (NTP 
2014). Patients with drug-susceptible TB receive anti-
TB treatment for at least six months while patients with 
MDR-TB and RR-TB receive longer treatments com-
prising of second-line drug regimens (NTP 2015). The 
sputum smear microscopy is used as an initial screen-
ing test for TB diagnosis, while GeneXpert assays are 
employed for the rapid detection of RR-TB at the dis-
trict level (NTP 2015). In the current study, we analyzed 
the prevalence of MDR-TB among different lineages 
prevalent in the Khyber Pakhtunkhwa (KPK) province 
of Pakistan. MDR-TB is notified after a confirmatory 
DST test performed at the central BSL-III laboratory.
Experimental
Material and Methods
Study site. Random samples were collected from 
25 districts of KPK using a simple random sampling 
(SRS) formula, which was previously used in a national 
TB survey of Pakistan:
where
SRS – Sample Random Sampling
N – total number of new smear positive cases reg- 
  istered in the lab
z – z-value (from the standard normal distribution) 
  that corresponds to the desired confidence level 
d – absolute precision 
p – expected proportion of MDR patient in the 
  target population
Study participants and sample collection. A total of 
1969 positive M. tuberculosis cases were calculated using 
this SRS formula. To achieve the target samples a total 
of 5759 clinical samples were collected from 25 districts 
and were diagnosed for TB. The patients’ data were col-
lected from their parents or the next caretakers.
Sputum processing. All received samples were 
digested and decontaminated using standard N-acetyl-
L-cysteine sodium hydroxide (NALC-NaOH) method 
(GLI 2014) in a biosafety level 3 laboratory (BSLIII) 
at the Provincial TB Reference Laboratory, Peshawar. 
Briefly, one aliquot was inoculated on the Lowen- 
stein Jensen medium (LJ) and in a Mycobacterium 
growth indicator tube (MGIT). Positive growth in the 
tubes was confirmed by Tbc ID device (Ref: 245159, 
Becton, Dickinson).
Drug susceptibility testing (DST). All confirmed 
mycobacterial isolates were processed for both pheno-
typic DST and molecular resistance assay. DST was per-
formed using a BD BACTEC MGIT 960 SIRE kit (Ref: 
245123, Becton, Dickinson), in which the final drug 
concentration was 1 μg/ml for RIF, and 0.1 μg/ml for 
INH. One sample aliquot was processed for acid-fast 
bacilli (AFB) microscopy using Primostar-LED fluo-
rescent microscopy.
Data analysis. Results were recorded in the local 
laboratory management information software and 
analyzed using SPSS V.15 (IBM, USA). Sensitivity and 
specificity were calculated using Medcalc  software 
(https://www.medcalc.org).
Results
Among 5759 TB suspects, 1969 (34%) were cul-
ture-positive, 3121 (54%) were culture-negative, and 
344 (6%) were contaminated. The proportion of TB 
was higher in females (39%) than males (29%), thus, 
a strong association was observed between the gender 
and tuberculosis disease (χ (3) = 68.2, p = 0.001). It was 
observed that the age group of 25–34 years was more 
likely infected with TB (40%) when compared to other 
groups (Table I). The susceptibility testing towards the 
first-line drugs as rifampicin, isoniazid, ethambutol, 
streptomycin, and pyrazinamide was performed on 
1969 culture-positive isolates.
DST results of 1969 isolates showed that 238 (12%) 
isolates were resistant to at least one drug, while 
97 (4.9%) were confirmed to be MDR-TB. The remain-
ing 1731 (88%) isolates were sensitive to all drugs. The 
drug resistance was the highest to streptomycin in 
173 (9%) isolates, followed by isoniazid in 119 (6%), 
ethambutol in 101  (5%), rifampicin in 99  (5%), and 
pyrazinamide in 65 (3%) isolates.
The drug resistance found was correlated with dif-
ferent factors from the patient history including age, 
gender, and treatment history. MDR was observed in 
61 (5.2%) males and 36 (4.5%) in female patients. No 
significant association of MDR with gender (χ (1) = 1, 
p-value = 0.26) or age group (χ (5.8) = 6, p-value = 0.44) 
was observed. The prevalence of MDR was higher in the 
age group of 55–64 years (6.4%), followed by a group of 
15–24 years (6%) (Table II). MDR correlation with pul-
monary and extra-pulmonary TB was also analyzed and 
it was found that the prevalence of MDR in pulmonary 
n (SRS) = 
N × z2 × p × (1 – p)
d2 × (N – 1) + z2 × p × (1 – p)
Prevalence of MDRTB in Khyber Pakhtunkhwa Pakistan2 135
TB was significantly higher 94 (5.3%) when compared to 
extra-pulmonary TB (1.5%), χ (5.3) = 1, p-value = 0.009. 
The resistance of the MTB isolates from the previously 
treated patients was significantly higher in 48 (10.4%) 
cases when compared to 49 (3.2%) untreated patients. 
It can indicate an association of drug resistance with 
the patient treatment history (χ (16) = 2, p-value = 0.001).
Discussion
MDR-TB is a major threat to public health. Moni-
toring its trends over time is crucial to prevent further 
emergence of drug resistance. Surveillance of drug 
resistance is, therefore, a critical component of any 
TB control Programme (Zignol et al. 2016). A  dec-
ade back, only 18 422 laboratory-confirmed MDR-TB 
cases were reported from 104 countries. It escalated 
to an estimated 490 000 cases in 2016 (WHO 2019). 
Even today MDR-TB is a persistent threat to the global 
community but unfortunately, only 47% of MDR cases 
could be diagnosed among the global estimates due to 
limited resources and laboratory gaps. Similarly, among 
all the registered MDR-TB cases, only 54% could be 
successfully treated. This study provides preliminary 
data of MDR-TB in KPK, which contributes to 13% of 
the national TB burden. In this first large-scale data, 
we found that MDR-TB was detected among 4.9% of 
Gender
Male 3189  947 (29%) 
χ (3) = 68.2, p = 0.005
Female 2570 1022 (39%)
Age group
01–14 437   96 (22%)
15–24 1137  396 (34%)
25–34 1180  473 (40%)
35–44 827  298 (26%)
45–54 732  227 (31%)
55–64 819  282 (34.4%)
65–100 627  197 (31.4%)
Treatment history
Previously Treated 1024  461 (45%) 
χ (3) = 68, p-value = 0.05
Never Treated 3922 1508 (38%)
Disease type
Pulmonary 5290 1864 (35%) 
χ (3) = 68, p-value < 0.05
Extra Pulmonary 469  105 (22%)
Sample type
Ascetic Fluid 91   10 (11%)
BAL* 172   45 (26%)
Bone 20    2 (10%)
CSF** 44    2 (5%)
Gastric Lavage 68    5 (7%)
Lymph Node 10    3 (30%)
Pericardial fluid 26    3 (12%) χ (36) = 259.6, p-value ≤ 0.05
Pleural Fluid 172   26 (15%)
Pus 54    9 (17%)
Sputum 5033 1858 (37%)
Synovial Fluid 3    0 (0%)
Tissue 20    5 (25%)
Urine 46    1 (2%)
Table I
Association of TB with gender, different age groups and treatment history, p-value
of < 0.05 shows a statistical significance.
* Bronchoscopy alveolar lavages,  ** Cerebrospinal fluid
Character TB Suspects Positive cases p-value
Ali S. et al. 2136
newly diagnosed patients. This figure is consistent with 
the first national DRS of Pakistan where it has been 
reported 3.7% MDR-TB cases (Tahseen et al. 2016) 
and 3.6% the global estimates (WHO 2019). Similar 
findings from Pakistan reported a 2–5% MDR-TB ratio 
(Javaid et al. 2008; Ejaz et al. 2010). A comparative high 
ratio of 29% and 9% MDR-TB was reported in early 
 literature from other areas of Pakistan (Javaid et al. 
2016; Shah et al. 2016). However, Akhtar et al. dem-
onstrated a much higher MDR ratio of 69% in a study 
performed in Punjab (Akhtar et al. 2016). Possible dif-
ferences in these reports might be due to the variance in 
study design and sample inclusion criteria. People ages 
15 to 34 years old were at high risk to develop MDR-TB 
(Hoa et al. 2015; Akhtar et al. 2016; Khan et al. 2018). 
The increased drug resistance in previously treated 
cases (10.4%) was high as compared to newly diagnosed 
patients. These findings are consistent with previously 
published data (Tahseen et al. 2016). TB has been found 
to be more prevalent in males (Neyrolles and Quintana-
Murci 2009); however, we did not detect a significant 
correlation of MDR-TB with gender, extra-pulmonary 
TB or a  sample type (Bhattacherjee and Datta 2014; 
Wattal et al. 2015). In contrast to this, data from Africa 
shows a relatively high prevalence of drug-resistant TB 
in women (O’Donnell et al. 2011).
In conclusion, MDR-TB is an emerging problem in 
Khyber Pakhtunkhwa, Pakistan. This study has high-
lighted the MDR surveillance among the population 
of a geographically distinct area of Pakistan. Knowing 
the approximate magnitude of MDR-TB, this study will 
help for better management of drug resistance towards 
global TB control 2030.
   ORCID
Anwar Sheed Khan https://orcid.org/0000-0003-0339-2487
Acknowledgements
We are thankful for the technical support of the Project Direc-
tor, Dr. Qasim Abbas for his technical support in this study.
Conflict of interest
The authors do not report any financial or personal connections 
with other persons or organizations, which might negatively affect 
the contents of this publication and/or claim authorship rights to 
this publication.
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Prevalence of Multi-Drug Resistant Mycobacterium Tuberculosis in Khyber Pakhtunkhwa – A High Tuberculosis Endemic Area of Pakistan

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