PREVALENCE OF MULTI-DRUG RESISTANT TUBERCULOSIS AMONG

TUBERCULOSIS PATIENTS IN PUNJAB

BS in Medical Laboratory Technology

Session 2019-2023

Submitted by

Muhammad Ayoub Ayesha Ehsan

HESC19115071 HESC19115033

SUPERVISORS

Dr. Ghulam Mustafa Kamal

Ms. Aiman Noreen

Institute of Health Sciences

Faculty of Food, Health Sciences and Technology

Khawaja Fareed University of Engineering and Information

Technology, Rahim Yar Khan

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PREVALENCE OF MULTI-DRUG RESISTANT TUBERCULOSIS AMONG

TUBERCULOSIS PATIENTS IN PUNJAB

Name: Muhammad Ayoub

Registration No. HESC19115071
Name: Ayesha Ehsan
Registration No. HESC19115033

A thesis submitted in partial fulfilment of the requirement for degree of

BS-Medical Laboratory Technology

Supervisor : Dr. Ghulam Mustafa Kamal

Co-supervisor : Ms. Aiman Noreen

Institute of Health Sciences

Faculty of Food, Health Sciences and Technology

Khawaja Fareed University of Engineering and Information

Technology, Rahim Yar Khan

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 Declaration

We Muhammad Ayoub and Ayesha Ehsan hereby state that my BS thesis titled
“Prevalence of Anemia and Associated Factors in Pregnant Women in South Punjab, Pakistan.”
is our own work and has not been submitted previously by us for taking any degree from Khwaja
Fareed University of Engineering and Information Technology, Rahim Yar Khan or anywhere
else in the country/world.
We solemnly declare that research work presented in the thesis is solely our research
work with no significant contribution from any other person. Small contribution/help wherever
taken has been duly acknowledged and that complete thesis has been written by us.
At any time if our statement is found to be incorrect even after our graduation the
university has the right to withdraw our BS degree.

Rahim Yar Khan, on June, 2023

Muhammad Ayoub (HESC19115071): Signature: __________________

Ayesha Ehsan (HESC19115033): Signature: __________________

Institute of Health Science

Research Certificate

3
We the undersigned certify that Muhammad Ayoub & Ayesha Ehsan candidate for the degree
of BS-Medical Lab Technology presented their research project of the following title
Prevalence of Multi-Drug Resistant Tuberculosis Among Tuberculosis Patients in Punjab,
Pakistan, as it appears on the title page and front cover of the research project. That the research
project is acceptable in form and content and displays a satisfactory knowledge of the field of
study.

Co-Supervisor Ms. Aiman Noreen Signature______________

Supervisor Dr. Ghulam Mustafa Kamal Signature______________

Deputy Director Dr. Ahmad Bilal Arif

Signature_______________
Examiner

Additional Director Dr Shabbir Hussain

Signature_______________

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 Acknowledgments

Thanks to the Almighty ALLAH, for granting knowledge and for all the blessings that he
has provided and poured upon us. Almighty Allah has shown unconditional and pure love by
using the people around us who are able to let us feel that we are loved and cared. We gratefully
acknowledge the support and patience of our family, school teachers, college teachers, professors
and friends throughout our studies and without them this project report could never have been
completed. We are grateful to Dr. Ghulam Mustafa Kamal & Ms. Aiman Noreen the supervisor
and co-supervisor of our project respectively for their support, guideline and great supervision.
We would also like to thank our institute, Khawaja Fareed University of Engineering and
Information Technology (KFUEIT) for providing us a platform where we were able to sharpen
our skills and develop as the individuals who can have a chance to grow in this ever-changing
environment.

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 ABSTRACT

Background and Introduction: Tuberculosis (TB) is an infectious disease usually

caused by Mycobacterium tuberculosis (MTB) bacteria. Tuberculosis generally affects the lungs,
but it can also affect other parts of the body. Most infections show no symptoms, in which case it
is known as latent tuberculosis. : The multidrug resistant tuberculosis epidemic is a main health
issue worldwide. Tuberculosis is a highly infectious disease, which considers major public health
issues around the world caused by bacteria known as mycobacterium. As a contagious disease.
Objective: The objective of the study is to see the prevalence of multi-drug resistant tuberculosis
among tuberculosis patients in south Punjab. Duration: Duration of the study was 2-3 months
till this thesis report. RESEARCH METHODOLOGY: Our mode of research is observational
Cross-Sectional study. DURATION: Duration of study is 3-4 months from the approval of
synopsis SAMPLE SIZE 50-60 Co-operative Respondents will be included. SAMPLE
SELECTION: Both male and female from 20 to 60 years’ INCLUSIONS CRITERIA: Both
male and female from 18 to 55 years’ age. Includes respondents from rural and urban areas.
EXCLUSION CRITERIA: Non-co-operative respondents will be excluded. DISCUSSION:
The analysis of the dataset consisting of 52 TB patients provides valuable insights into the
prevalence of drug resistance in this population. The mean and standard deviation values indicate
variability in the test results. The frequency distribution shows a significant proportion of
positive TB cases. The levels of MTB detected vary, but further drug susceptibility testing is
needed to determine drug resistance.
Key words:

6
Abbreviations and acronyms

MDR-TB: MULTI DRUG RESISTANT-TB

.
TAB: Testing for TB antibodies

FMDR-TB: Frequency of MDR-TB

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Contents
DECLARATION.............................................................................................................................3
ACKNOWLEDGMENTS...............................................................................................................5
ABSTRACT....................................................................................................................................6
CHAPTER 1 INTRODUCTION........................................................................12
1.1 BACKGROUND....................................................................................................................................................12
1.2 INTRODUCTION..................................................................................................................................................12

CHAPTER 2 LITERATURE REVIEW............................................................14

CHAPTER 3 HYPOTHESIS AND PROBLEM STATEMENT.................................17
3.1 HYPOTHESIS.......................................................................................................................................................17
3.2 PROBLEM STATEMENT......................................................................................................................................17

CHAPTER 4 OBJECTIVE...............................................................19
OBJECTIVE...............................................................................................................................................................19

CHAPTER 5 OPERATIONAL DEFINITIONS................................................20

5.1 ANEMIA..............................................................................................................................................................20
5.2 TYPES OF ANEMIA.............................................................................................................................................20
5.2.1 Microcytic anemia......................................................................................................................................20
5.2.2 Macrocytic anemia.....................................................................................................................................21

CHAPTER 6 RESEARCH METHODOLOGY.............................................22

6.1 DURATION..........................................................................................................................................................22
6.2 SAMPLE SIZE......................................................................................................................................................22
6.3 SAMPLE SELECTION..........................................................................................................................................22
6.4 INCLUSION..........................................................................................................................................................22
6.5 EXCLUSIONS.......................................................................................................................................................23
6.6 DATA COLLECTION PROCEDURE......................................................................................................................23
6.7 STUDY DESIGN AND PARTICIPANTS...................................................................................................................23
6.8 DATA ANALYSIS PROCEDURE............................................................................................................................23
6.9 ETHICAL CONSIDERATIONS..............................................................................................................................23

CHAPTER 7 RESULTS AND STATISTICAL ANALYSIS.........................................24

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 7.1 RESULTS.............................................................................................................................................................24
7.2 TABLES...............................................................................................................................................................24

CHAPTER 8 DISCUSSION...................................................................34
CHAPTER 9 CONCLUSION....................................................................38
CHAPTER 10 REFRENCES..................................................................39

List of Tables

Table 7.3.1……………………………………………………………………………………..24
Table 7.3.2……………………………………………………………………………………..25
Table 7.3.3……………………………………………………………………………………..26
Table 7.3.4……………………………………………………………………………………..27
Table 7.3.5……………………………………………………………………………………..28
Table 7.3.6……………………………………………………………………………………..29

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List of Figures

Fig.7.3.1……………………………………………………………………………………….30
Fig.7.3.2……………………………………………………………………………………….31
Fig.7.3.3……………………………………………………………………………………….32
Fig.7.3.4……………………………………………………………………………………….33

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Chapter 1 INTRODUCTION

1.1 Background
Tuberculosis (TB) is an infectious disease usually caused by Mycobacterium
tuberculosis (MTB) bacteria. Tuberculosis generally affects the lungs, but it can also affect other
parts of the body. Most infections show no symptoms, in which case it is known as latent
tuberculosis. Around 10% of latent infections progress to active disease, which, if left untreated,
kill about half of those, affected.

1.2 Introduction

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 The multidrug resistant tuberculosis epidemic is a main health issue worldwide.
Tuberculosis is a highly infectious disease which considers major public health issues
around the world caused by a bacteria known as mycobacterium. As a contagious disease.
According to our research by World Health Organization in 2016, the mortality rate what's
5 per thousand people in 2000 but it kept increasing and now its 370 cases out of 1000
people. This report shows that the health issue of people was a major concern of diabetes
mellitus and tuberculosis according to the current prevalence. This huge increase in this
disease was probably contributed by the prevalence of undiagnosed diseases. There was the
need of an effective approach you are such diseases and use super sensitive tests to prevent
such diseases. In addition to the management of patients dealing with such contagious
diseases, this has to be treated with anti-tuberculosis medication questions with the clinical
experiences in the potential between DM medicines and different factors(Cho, Yoon et al.
2021) .
The multidrug-resistant tuberculosis (MDR-TB) epidemic has become a major
worldwide health issue. Prior anti-tuberculosis medication is the main risk factor for the
development of MDR-TB.(Du, Zhang et al. 2021).Multidrug-resistant tuberculosis (MDR-
TB) is becoming more commonplace globally. The most significant risk factor for
developing MDR-TB is prior TB therapy; individuals who have never had treatment are
still at risk because of spontaneous mutations or the spread of drug-resistant strains (Ejaz,
Siddiqui et al. 2010).

Pakistan is also thinking about using MDR second-line therapy. Thus, it is crucial to
gather fundamental data in common occurrence of multi drug resistance in the nation. Many
variables are acknowledged as causing MDR-TB to develop and spread across society on a
global scale.(Khursheed, Asif et al. 2022) multidrug-resistant tuberculosis is a serious public
health issue that affects many nations, including Malaysia. It is particularly prevalent in
Southeast Asia and continues to challenge efforts to control the disease globally. 26.168 cases of
TB were reported in Malaysia in 2016; this is an 8% increase from the 24.2 cases were reported
in 2015. Moreover, the mortality rate from tuberculosis enormously increased from 5.5% to 6.5%
per 100,000 individuals in 2015 (Molla, Reta et al. 2022).
The most recent reported fatal disease globally is tuberculosis (TB), which is brought on
by the bacteria Mycobacterium tuberculosis. The potent first-line medications, are the examples

12
of multidrug-resistant tuberculosis. A significant global problem in tuberculosis (TB) control is
multidrug resistant tuberculosis was predicted to have affected 484,000 persons worldwide in
2018, including about 130,000 people in India. Treatment outcomes for people with MDR-TB
remain dismal despite significant therapeutic improvements over the past ten years. Although the
patient's pharmacological regimen may contribute to some of the variation in treatment outcomes
(Sambas, Rabbani et al. 2020).
MDR-TB is defined by expensive medication, prolonged therapy, and subpar efficacy
when compared to other pharmaceuticals.(Du et al., 2021) Inadequate TB patient health
education, an increase in respiratory infections, and several other potential health concerns
associated with this disease all contribute to the MDR-TB epidemic. Incidence of MDR-TB
(Thomas et al 2021). Due to patients' ignorance of the disease's causes and lack of awareness of
its risk factors, the number of cases is anticipated to rise in 2021.(Sharma, Kumar et al. 2011).
The success rate of drug resistance worldwide is low, and the causes of unsuccessful treatments
are not well understood. Moreover, available by combining mortality, and to follow-up into a
category, investigations are concentrating on identifying predictors of unsatisfactory treatment
outcomes. Yet, this might mask the true risk factors for dying and not responding to treatment
(Thomas, Kumar et al. 2021) .
The immune system of the infected person fights off the TB organisms, and in the
majority of cases, the infection is lifelong and does not reactivate.(Harichander, Wiafe et al.
2022).First-line medication resistance significantly decreased in Northern Taiwan from 2000 to
2006 (P 0.001) and in Central Taiwan from 2003 to 2007, according to research conducted in
hospitals.(Wu, Hsiao et al. 2023).A report from the Office of Foreign Workers Administration
states that in 2015, 12,334 and 2801 foreign workers, respectively, were employed in Chiang Rai
and Chiang Mai (Saikaew, Thongprachum et al. 2022).

A report from the Office of Foreign Workers Administration states that in 2015, 12,334
and 2801 foreign workers, respectively, were employed in Chiang Rai and Chiang Mai (Prakash,
Kumar et al. 2016).

Drug-resistant TB is primarily a man-made phenomenon, according to WHO (2006), and

is primarily caused by patient non-compliance, inadequate supply and inappropriate
administration of drug regimens, inadequacy to monitor treatment, and most importantly, the

13
presence of poorly organized TB control programmes. MDRTB has microbial, clinical, and
programmatic causes as well (Aminu and Tukur 2016).

A total of 352 cases of MDR-TB were reported in 2018, including 182 new cases (3.5%
more than the 3.1% (170) previously treated patients) (Rajendran, Zaki et al. 2020).

The microbiological confirmation of MDR-TB is based on labor-intensive, expensive,

and time-consuming drug susceptibility testing (DST) and culture procedures, which call for a
significant investment in laboratory infrastructure and are not frequently used in nations with
scarce resources (Baya, Achenbach et al. 2019).

This is an increase over the prior year, when there were reportedly 580,000 drug-
resistant TB cases, 100,000 of which were rifampicin-resistant. According to estimates, 250,000
people died from MDR-TB in the same year, predominantly in Asia (Okethwangu, Birungi et al.
2019).

With 44% and 24%, respectively, of all new tuberculosis cases and deaths, Southeast Asia
and Africa are the most frequently reported regions (Iradukunda, Ndayishimiye et al. 2021).

Although a few models have predicted the prevalence of TB in various countries, few
have provided a precise estimation for MDR-TB (Li, Shi et al. 2020).

Multidrug-resistant tuberculosis (MDR TB), which is defined as bacilli resistant to both

isoniazid and rifampicin with or without the participation of other medications, may develop as a
result of treatment failure (Sambas, Rabbani et al. 2020).

While primary resistance can be controlled with treatments to stop transmission, acquired
resistance can be avoided by ensuring adherence to optimal medication (Khan, Yates et al. 2019).

In addition, a newly released meta-analysis study of individual patient data revealed that
61% of MDR-TB patients had successful treatment (Tola, Holakouie-Naieni et al. 2021).

Africa has a very high incidence rate of the disease and is home to 25% of all cases
worldwide, where subpar diagnosis and treatment are extremely widespread. With an expected
29,000 cases in 2019 in Sudan, TB is one of the primary public health challenges in
underdeveloped nations (Hajissa, Marzan et al. 2021).

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 The MDR-TB outbreak needs to be controlled immediately. Bhutan has little information
on the variables influencing MDR-TB risk. According to a descriptive study that included 19
MDR-TB patients from a treatment facility in Bhutan, the majority of the cases did not follow
the DOT method, had a history of TB treatment, and had contact with MDR-TB cases (Tenzin,
Chansatitporn et al. 2020).

15
Chapter 2 LITERATURE REVIEW

A study by Mahindra Rajendran published on August 6th, 2020, investigated the

prevalence of MDR-TB among tuberculosis patients in Malaysia. The study used data from the
Malaysian National Tuberculosis Information System (TBIS) from 2009 to 2019. The study
identified 989 patients with MDR-TB and recorded their demographic information, including
marital status, gender, ethnicity, employment situation, drinking habits, diabetic condition, and
smoking history. Statistical analysis was conducted using SPSS software version 20. The study
found an overall prevalence of MDR-TB among TB patients in Malaysia of 0.34%. The results
also revealed significant differences in MDR-TB incidence between male and female patients
(0.44% vs 0.20%, p=0.001), single and married patients (1.63% vs 0.24%, p=0.001), and among
patients from different ethnic backgrounds(Karimi-Maleh, Karimi et al. 2020).

A study by Kindu Alem Molla published on June 30, 2022, focused on the alarming
spread of multidrug-resistant TB worldwide, particularly in East Africa, where it is emerging as a
public health issue. The study aimed to assess the prevalence of multidrug-resistant TB among
pulmonary tuberculosis-positive individuals in the region. A systematic search was conducted
using six electronic databases, including PubMed, EMBASE, Scopus, Science Direct, Web of
Science, and Google Scholar, for published English-language articles. The random effects model
developed by Der Simonian and Laird was used to calculate the combined prevalence of
multidrug-resistant TB and related risk factors, and publication bias was evaluated using Egger’s
regression asymmetry test and big rank correlation methods. The study included 16 articles
published between 2007 and 2019, out of 1025 articles with recognized citations, and utilized
STATA 14 software. The meta-analysis revealed a combined prevalence of multidrug-resistant
TB of 4% (95% CI = 2-5%) among patients with tuberculosis who had previously received
treatment and 21% (95% CI: 14–28%) among newly diagnosed cases. The study identified
various risk factors for multidrug-resistant TB, including resident circumstances lifestyle
variables (such as drinking, smoking, and substance abuse), past health conditions, a history of
diabetes, and an infection with the human immunodeficiency virus (Molla, Reta et al. 2022).

Globally, TB is a serious public health issue. Multidrug-resistant (MDR) TB's

introduction has made the situation even more difficult by resulting in subpar treatment results
and higher treatment expenditures for patients and healthcare systems. In order to ascertain the

16
prevalence of MDR-TB and associated risk factors among TB patients in Makkah, this study set
out to identify these variables. Patients in the research had an average age of 43.4 18.7 years, and
66.5% of them were men. Around 40% of patients had chronic illnesses, and 5% had lung
problems other than TB. Extrapulmonary infections affected about 13% of the patients.
Streptomycin (25.9%) and isoniazid (11.1%) were the most often impacted drugs in resistant
cases, according to the research, which indicated that 17.1% of TB patients developed treatment
resistance. About 5% of TB patients, MDR-TB was identified. Age, smoking, lung conditions,
and prior TB were all significantly linked to MDR-TB(Sambas, Vaidyanathan et al. 2020).

In recent years, the incidence of multidrug-resistant (MDR) tuberculosis (TB) has

significantly increased worldwide. It has been estimated that around 30,000 pediatric TB cases
annually, equivalent to 3% of all pediatric TB cases, are MDR. While most children with MDR-
TB can be successfully treated, those with extensively drug-resistant (XDR) TB had limited
treatment options and no standard regimen until five years ago. This publication aims to discuss
the current understanding of treating MDR- and XDR-TB in children, with a particular focus on
the characteristics and available data on the use of two promising new drugs, bed aquiline and
delamanid.The studies included in this review were obtained from the PubMed database
(Khabarova, Malandraki et al. 2021).

In a recent retrospective study, Khursheed investigated the susceptibility pattern of anti-

TB drugs over a period of five years at Indus Hospital and Health Network. Out of 20,014
samples tested, 23.1% were found to be MTB positive. DST was performed on 95.9% of the
isolates using BACTEC MGIT, and it was observed that 52% of the isolates were from males
and 48% from females. The study found no significant relationship between gender and the
likelihood of having MDR-TB. The highest rate of isolation was observed among previously
treated patients and those aged 25–55 years (62% vs. 36%). Among the MTB positive cases,
91.5% were pulmonary and 8.5% were extra pulmonary. The study revealed high resistance to
isoniazid (58% in pulmonary, 12.7% in extra pulmonary), rifampicin (58.7% in pulmonary, 8.2%
in extra pulmonary), and levofloxacin (29% in pulmonary, 20% in extra pulmonary) antibiotics
(Khursheed, Asif et al. 2022).

Kateete conducted a study to determine the resistance pattern of second-line anti-TB

drugs, the presence of quinolone resistance-determining regions (QRDRs) in gyrA and gyrB

17
genes, and the drug resistance-associated rrs gene in 80 TB isolates from Uganda and Somalia
between 2014 and 2016. Of the 80 isolates, 40 were identified as MDR-TB, of which 28 (70%)
were resistant to second-line anti-TB injectable drugs, 18 (45%) were resistant to levofloxacin,
and 12 (30%) were extensively drug-resistant (XDR-TB). The remaining 40 isolates were
susceptible to MDR-TB. Out of the 40 second-generation drug-resistant isolates, 38 were
subculture. The study detected gyrA resistance mutations in 72.2% and gyrB resistance
mutations in 22.2% of the levofloxacin-resistant MDR-TB isolates. Overall, drug resistance
mutations in gyrA and gyrB occurred in 77.8% (14/18) of MDR-TB and levofloxacin-resistant
isolates. Drug resistance mutations in the rrs gene occurred in 64.3% (18/28) of MDR-TB
isolates resistant to second-line injectable drugs (Kateete et al 2019).

Al-Mutairi conducted a study on 152 MTB isolates collected between 2006 and 2019.
Out of the 152 isolates, 50 were susceptible to anti-TB drugs, while 102 were identified as MDR-
TB. Among the 102 MDR-TB isolates, 78 were pulmonary and 24 were extra-pulmonary,
whereas out of the 50 susceptible isolates, 34 were pulmonary and 16 were extra-pulmonary. All
the isolates were cultured from newly diagnosed TB patients before initiating the anti-TB
treatment. The Accu Probe DNA assay and multiplex PCR assay were used to identify all the
isolates. Among the 102 MDR strains tested by the gMTBDRslv1 and gMTBDRslv2 assay, 12
isolates contained a mutation in gyrA, while 90 isolates had wild-type gyrA. Eight isolates were
found to have a mutation in rrs; 59 isolates had the embB mutation, two had the gyrB mutation,
and one isolate had the eis mutation (Koo, Kang et al. 2021).

The present study will present a patient with drug resistant tuberculosis along with
underlining medications and currently receiving such precautions and treatments to control for
next few months. This report presents the tasks in the management of such contagious diseases
and conditions. Pakistan has a prevalence rate of 263/100,000 and an incidence rate of
181/100,000. Limited information has been given in the ways of medication resistance in the
nation, despite the significant illness burden. According to a WHO study from 2008, Pakistan's
MDR rates for untreated and previously treated TB patients are respectively 3.4% and 36%
(Kateete, Kamulegeya et al. 2019).

It was shown that certain sociodemographic risk variables, such as monthly income,
patient residency, and family size, may increase the likelihood of MDR-TB infections in TB

18
patients.(Rajendran, Zaki et al. 2020) More people die from tuberculosis (TB) each year than
from any other bacterial infection. A survey predicted 10 million cases of tuberculosis in 2018.
The prevalence of sickness varies greatly between nations, with an annual average of 100,000
people worldwide. In the meantime, the mortality among HIV-negative individuals was expected
to be 1.2 million (range 1.1–1.3 million) in 2018 and among HIV-positive individuals, 251,000
(range 223,000–281,000). In 2018, 11% of cases of TB involved children, with rates in
prospering nations are higher (Rajendran, Zaki et al. 2020).

A total of 16 publications were included in the final meta-analysis out of the 1025 articles
with recognized citations. In patients with tuberculosis who had previously received treatment as
well as newly diagnosed cases, the combined prevalence of multidrug-resistant tuberculosis was
4% (95% CI = 2-5%) and 21% (95% CI: 14-28%), respectively. The greater frequency of
multidrug-resistant tuberculosis in East Africa was caused by risk variables including residence
circumstances, lifestyles (including smoking, drinking, and abusing drugs), prior medical history,
diabetes history, and human immunodeficiency virus infection. The systematic search of the six
electronic databases yielded a total of 1,025 articles. After screening and selection, 16 articles
were included in the final meta-analysis. These articles provided insights into the prevalence of
multidrug-resistant tuberculosis (MDR-TB) among pulmonary tuberculosis-positive individuals
in East Africa (Molla, Reta et al. 2022).

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Chapter 3 HYPOTHESIS AND PROBLEM STATEMENT

3.1 Hypothesis
In TB patients testify either there is multidrug resistance in Mycobacterium tuberculosis
bacilli in TB infected patients or not.

The results of the present study may be extremely important in helping medical
professionals and policymakers create and put into practise efficient approaches for the
prevention and treatment of MDR-TB among TB patients who have tested positive for TAB.
Moreover, it can aid in efforts to achieve the Sustainable Development Goal of ending the TB
pandemic and lowering the burden of MDR-TB worldwide.

.
3.2 Problem Statement
The issue at hand is the high prevalence of multi-drug resistant tuberculosis (MDR-TB)
among TB patients in the Punjab state. MDR-TB refers to Mycobacterium tuberculosis strains
that are resistant to at least two of the most powerful first-line anti-TB medications, isoniazid and
rifampicin. MDR-TB has emerged and expanded, posing considerable obstacles to TB control
programs and public health efforts.

The goal of the current study is to determine the prevalence of MDR-TB among TB
patients in Punjab and to identify the factors that contribute to its occurrence. The study's goal is
to figure out how many MDR-TB cases there are among newly diagnosed and previously treated
TB patients in different parts of Punjab. It also intends to look into the risk factors linked with
the development-TB symptoms include poor treatment adherence, prior anti-TB drug exposure,
and comorbidities such as HIV/AIDS.

MDR-TB incidence in Punjab has serious implications for the efficiency of TB treatment
and control initiatives. Understanding the scope of MDR-TB in this region would aid healthcare
authorities and policymakers in developing targeted interventions and strategies to address
medication resistance concerns. The findings of the study will help to shape evidence-based
policies, recommendations, and initiatives aimed at reducing the burden of MDR-TB and
improving treatment outcomes among TB patients in Punjab.

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Chapter 4 OBJECTIVE

Objective
Main objective of our study is:
• To study about prevalence of multidrug-resistant tuberculosis (MDR-TB) among TB
patients who have been diagnosed using tuberculosis antibody (TAB) testing

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Chapter 5 OPERATIONAL DEFINITIONS

5.1 Anemia
Anemia is a medical condition marked by a lack of red blood cells or hemoglobin in the
blood. A protein named hemoglobin is found in red cells(RBC), is in charge of carrying oxygen
throughout the body. Numerous factors, including insufficient red blood cell synthesis, increased
red blood cell oxidation, and excessive bleeding, can contribute to anemia. The most typical
signs of anemia include weakness, exhaustion, shortness of breath, pale complexion, and vertigo.
A diet change, medication, or blood transfusions may be used to treat anemia, depending on the
underlying reason.
5.2 Types of Anemia
5.2.1 Microcytic anemia is a kind of anemia that is specified by very small RBCs.
There are numerous reasons why this might happen, including:
• Iron deficiency anemia: The most prevalent reason for microcytic anemia is
this. It occurs when the body cannot produce enough hemoglobin, the protein
that transport oxygen in red blood cells, due to insufficiency of iron in the body
• Thalassemia: A genetic disorder of blood called thalassemia affects how much
hemoglobin is made. A thalassemia patient's defective hemoglobin might result
in the development of smaller red blood cells.
• Anemia of chronic disease: : Individuals with cancer, autoimmune disorders,
or long-term infections are susceptible to acquiring this type of anemia It is
believed to be brought on by inflammation and other elements that prevent the
body from producing red blood cells.
 Lead poisoning: Because lead interferes with the development of heme, a
substance
essential to the formation of hemoglobin, exposure to high levels of lead can result
in microcytic anemia.
 Sideroblastic anemia: When the bone marrow is unable to utilize iron to create
hemoglobin, a rare form of anemia called hemolytic anemia develops

22
5.2.2 Macrocytic anemia
A kind of anemia known as macrocytic anemia is characterized by unusually big red blood cells.
This can occur due to a number of different causes, including:

 Vitamin B12 deficiency anemia: The most frequent reason for macrocytic anemia is
this. It happens when the body is unable to produce enough healthy red blood cells due to
a lack of vitamin B12
 Folate deficiency anemia: : Another crucial vitamin for the creation of red blood cells is
folate. Macrocytic anemia can result from a folate deficiency.
 Liver disease: Some types of liver disease can affect the production of red blood cells,
leading to macrocytic anemia.
 Alcoholism: Chronic alcoholism can cause macrocytic anemia by interfering with the
absorption of nutrients like vitamin B12 and folate.
 Medications: Certain medications, such as chemotherapy drugs and some
anticonvulsants, can cause macrocytic anemia.

23
Chapter 6 RESEARCH METHODOLOGY

6.1 Duration
03 months after the approval of synopsis.

6.2 Sample Size

52 Co-operative respondents were included.

A commonly targeted level of confidence is 95%, and many researchers typically report
their findings using a 95% confidence interval (CI). However, researchers who desire greater
confidence may opt for a 99% confidence interval.(Pourhoseingholi et al., 2013)
6.3 Sample Selection
Mode of research is Observational/ Cross-Sectional.

6.4 Inclusion
• Both Male and Female Included

• Respondents from rural and urban areas will be included

• Respondents 12 to 81 years age group will be included

6.5 Exclusions
• Non cooperative respondents will be excluded

6.6 Data Collection Procedure

This cross-sectional study will be caried out in the public sector for three months after the
approval of synopsis. Patients will be recruited in the study keeping in mind the inclusion and
exclusion criteria. Written informed consent will be obtained from all subjects before and all
possible benefits and expected risks will be explained to participants. Basic demographic and
clinical information about risk factors and medical history will be obtained from patients and
noted down on a pre designed data collection sheet.
6.7 Study design and participants:

24
 This was a cross-sectional and observational study conducted at District Head Quarter
Teaching hospital Dera Ghazi Khan.The study includes in 52 participants. The residency of
participants is both rural and urban.

6.8 Data Analysis Procedure

Data is analyzed by latest version of statistical software named as SPSS 27.0 (Statistical
packages for social sciences). Basic descriptive statistics will be calculated (including median
and quartile, SD, max/min, percentile). Then different statistical tests applied to get results.

6.9 Ethical Considerations

i. Written informed consent (attached) will be taken from all the participants.
ii. All information and data collection will be kept confidential.
iii. Participants will remain anonymous throughout the study.
iv. The subjects will be informed about the disadvantages or risks of the procedure in the
study.
v. They will also be informed that they will be free to withdraw at any time during the
process of the study.
vi. There will great benefits to the participant that would result from their participation in
this research.
vii. We will do everything we can to protect your privacy. Your identity will not be revealed
in any publication resulting from this study.
viii. Your participation in this research study is voluntary. You may choose not to participate
and you may withdraw your consent to participate any time. You will not be penalized in
any way should you decide not you participate or to withdraw from this study.

25
Chapter 7 RESULTS AND STATISTICAL ANALYSIS

7.1 Results

There are no blood sample taken personally, the hemoglobin values are noted from the
reports of patients.
Statistical software was used to analyses the data collected., including the mean,
frequency, standard deviation, t-test, group statistic and other required tests. A p-value of less
than 0.05 was considered statistically significant.

7.2 Tables

Table 7.3.1 Mean and St. Deviation value of Hb%

N Mean Std. Deviation

Hemoglobin% 132 2.1288 .75569

Valid N 132
(listwise)

Table 7.3.1 based on the descriptive statistics it shows the result of Hb% of 132 patients which
are all pregnant. The mean value indicate that all the pregnant women are all most anemic. A few
patients are non-anemic. Standard Deviation show less variation it’s mean that there is no
variability in Hb% value in patients.

Table 7.3.2 Frequency and percentage of severity of anemia

26
 Hb% Frequency Percentage%
below 8 24 18.2
8.0-9.9 73 55.3
10-11.5 29 22.0
above 11.5 6 4.5
Total 132 100.0

Table 7.3.2 shows the frequency of severity of anemia as we divided severity of anemia in the
base of Hb% value which is shown as:

Severity of anemia Hb%

Mild 10.0-11.5

Moderate 8.00-9.9

Severe Below 8.00

According to table 1.2, out of 132, 24 patients are in severe condition of anemia, 73 patients are
in moderate phase of anemia, 29 patients have mild anemia and only 6 patients are normal. This
table show that anemia is common in pregnant women.
Table 7.3.2 also indicates that there are 95.45% patients are anemic and only 4.55% patients are
normal.

Table 7.3.3 Frequency of Iron Supplements

Frequency Percentage %

27
Yes 68 51.5
No 64 48.5
Total 132 100.0

Table 7.3.3 shows that out of 132 patients 68 (51.5%) patients take iron supplement to control
the iron deficiency anemia. Other patients may have any other type of anemia. Some normal
pregnant women also use iron supplements as prescribed by the doctor.

Table 7.3.4 Frequency of Balance Diet

Frequency Percent
Yes 76 57.6

28
No 56 42.4
Total 132 100.0

Table 7.3.4 shows that 57.6% patients take balance diet. Other 42.4% patients cannot take
normal diet. It causes anemia during pregnancy.

Table 7.3.5 Frequency of B12 or folic acid

Frequency Percent

29
Yes 28 21.2
No 104 78.8
Total 132 100.0

Table 7.3.5 indicates that 21.2% patients take B12 or folic acid supplements 78.8% patients
cannot take. It shows that mostly patients are of iron deficiency anemia who take iron
supplements.

Table 7.3.6 Association of different factors with Anemia

Factors Frequency Percentage

Yes No Yes No

30
 Education 45 87 34.1 65.90
Hypertension 33 99 25.0 75.0
Miscarriage 24 108 18.2 81.8
Exercise 43 89 32.6 67.4
Viral Infections 8 124 6.0 94.0
Abnormality in 17 115 12.9 87.1
menstrual cycle

Table 7.3.6 indicate the results of different factors which cause anemia during pregnancy.
According to table illiteracy, hypertension, miscarriage, no exercise, viral infections and
abnormality in menstrual cycle are the most common factors which cause anemia. Anemia and
levels of education in the current study, it was found that the percentage of anemia was higher in
illiterate people and lower in literate people.

31
 Figure7.3.1 Describing the severity of anemia

The given graph shows the severity of anemia . We divide severity of anemia into normal( Hb%
above 11.5), mild (Hb% 10.0-11.5), moderate( Hb% 8.0-9.9), severe ( Hb% below 8). Severity of
anemia along x-axis and number of patients along Y-axis.

32
Figure7.3.2 Indicates the education level of participants.

33
 Figure 7.3.3 Shows the age of different participants.

It shows that mostly patients are between 27-30 years old.

34
 Figure7.3.4 Determines the month of pregnancy

It shows that mostly women are in their second trimester of pregnancy.

Chapter 8 DISCUSSION

According to the current study, pregnant women had a higher percentage of anemia than
other groups. The percentage of anemia is 95.45%. The samples are calculated from both rural

35
and urban residency. According to the current study, anemia rates are higher in rural than urban
areas. However, some research indicate that anemia is more common among pregnant women
living in metropolitan areas. As contrast to our research and the present study, the prevalence of
anemia is observed to be high in some metropolitan areas of Pakistan, where it is reported to be
90.05% (Baig-Ansari et al., 2008).
In present study we calculated percentage of anemia on the basis of its severity. On this
base we divided anemia into three phases i.e., mild, moderate and severe. The current research
demonstrates that the moderate anemia is common among expectant mothers. The percentage of
mild anemia is 22.0%, moderate anemia is 55.3% and severe anemia is 18.2%.  In 2001, WHO
and another study have reported the moderate prevalence of anemia (33%) in Iran (Sadeghian et
al., 2013).
We found a lower relation between anemia and abnormality in menstrual cycle. Although
the menstrual cycle is not a primary determinant in the development of anemia, it may
exacerbate the condition in pregnant women.
Exercise as prescribed by doctor play important role in pregnancy. All most 67.4%
patients do not exercise daily as in our research and other also do not exercise regularly. In rural
area mostly women assume that daily house work is an exercise due to the lack of awareness and
illiteracy. But daily routine work is not a proper exercise. Proper exercise is that which is
prescribed by doctor during pregnancy. Due to these chances of anemia increase during
pregnancy. A systematic review was conducted that examined the relationship between exercise
and oxygen delivery during pregnancy. The review revealed that regular physical activity
improved maternal oxygen-carrying capacity, resulting in enhanced oxygen delivery to the
placenta. This improved oxygenation can positively impact fetal growth and development while
supporting the prevention and management of anemia (Serván-Mori et al., 2022).
Viral infections are the basic factors of anemia in any person. They cause anemia in
pregnant women because they cause blood deficiency. The present study shows that in 95.5%
anemic patients 6.0% patients have viral infection. Viral infection causes the severity of anemia.
Viral infections can contribute to an increased risk of anemia in pregnant women. Certain viral
pathogens, such as parvovirus B19 and cytomegalovirus (CMV), have been associated with the
development of anemia. The synthesis and survival of red blood cells are both directly impacted
by these viruses, which lowers hemoglobin levels.

36
 Studies have indicated that pregnant women infected with parvovirus B19 or CMV may
experience a lack of red blood cells production due to the viral invasion of bone marrow cells
responsible for erythropoiesis. This disruption in red blood cell synthesis can contribute to the
development of anemia during pregnancy (Hardy, 1965).
The hypertension is another factor which cause anemia during pregnancy. The present
study indicate that 25.0% pregnant women are victim of anemia. Both anemia and hypertension
combine to cause other diseases. Hypertension can negatively affect blood flow to various
organs, including the placenta, reducing oxygen delivery to the developing fetus. This
compromised oxygenation can contribute to the development of anemia or worsen existing
anemia. Additionally, hypertensive disorders can disrupt the normal functioning of the kidneys,
which play a vital role in red blood cell production and the maintenance of overall blood
composition (Milman, 2011).
Iron, B12, and folic acid deficiency are the other common causes of anemia in pregnant
women. Iron deficient women will see an exciting rise in their iron requirements throughout
pregnancy. If treatment is delayed in iron deficiency anemia, both the prevalence and the severity
of the condition may significantly rise when the ladies are pregnant.
The likelihood of anemia during pregnancy varies greatly by geographical area. Europe
and the Americas have the lowest frequency of iron deficiency anemia (25%) while South-East
Asia and Africa have the highest prevalence (48 and 57%, respectively). In Denmark, contrasting
to pregnant women who take 40 mg of iron supplements daily, who have anemia at a prevalence
of less than 5%, pregnant women who do not take iron supplements have a prevalence of anemia
of 25% (Milman, 2011).
Folic acid insufficiency is the other fundamental factor shortage that causes anemia.
Since folic acid deficiency anemia in many communities has not been as fully examined as iron
deficiency anemia or the usage of iron supplements up to this point, further study is necessary
before we can have a clearer picture of the incidence of folate insufficiency in numerous
populations. Megaloblastic anemia, which has a high mean cell volume, is a specific kind of
anemia caused by folic acid deficiency. According to estimates, up to 25% to 72% of pregnant
women in underdeveloped nations suffer from a folic acid shortage (de Benoist, 2008). Folic acid
deficiency is linked to a high risk of neural tube malformations and other organ problems in
fetuses and newborn newborns. The second most common vitamin deficiency that results in

37
anemia, a megaloblastic anemia with a high mean cell volume, is probably vitamin B12
deficiency. 40% of pregnant women, according to studies from Lebanon and Turkey, had vitamin
B12 deficiencies, which may be brought on by the pernicious anemia or the food cobalamin
malabsorption syndrome in addition to inadequate dietary vitamin B12 consumption. The lack of
vitamin B12 is a major issue in poor nations (Al Khatib et al., 2006).
We found that 51.5% patients take iron supplements and 21.2% patients take folic acid or
B12 supplements to control anemia. The rest patients cannot use these supplements so the risks
of anemia are high in them. These nutritional supplements are used to manage anemia. mostly
folic acid or B12 deficient anemia and iron deficiency anemia.

Miscarriage is also a factor which cause anemia in next pregnancy. In our research 18.2%
patients are found who have the previous history of miscarriage. These patients are anemic with
different severity of anemia. The impact of miscarriage on anemia risk lies in the potential
depletion of essential nutrients during the pregnancy loss. If the body does not fully recover from
the miscarriage or if proper nutrition is not maintained, it can lead to deficiencies that increase
the risk of anemia during subsequent pregnancies (O’Kelly et al., 2022).
The other most important factor is education. Illiteracy causes anemia because pregnant
women who are un educated do not know the factors or diet plan become the victim of anemia.
In this present study 65.9% patients are uneducated. So, they do not know the factors of anemia
or how to overcome anemia, therefore they became the victim of anemia. 34.1% patients are
educated they control anemia by different methods as they know the risk factors and knowledge
about anemia. The situation of anemia increases steadily with the decrease of education level.
Education is connected with anemia high the education low the anemia and vice versa.
Aurangabad City, India, pregnant women with primary, middle, and secondary education had
anemia rates of 96.4%, 94.8%, 92.1%, and 91.5%, respectively, according to a prior study. In
Jima town, Southwest Ethiopia, anemia prevalence among illiterate women was highest at 53.7%
compared to literate women's prevalence of 37.1% (Ullah et al., 2013).

38
Chapter 9 CONCLUSION

Among the current study, anemia was prevalent among pregnant women to a significant
degree (95.5%). The percentage was high in rural area as compared to the urban. We divided the
severity of anemia into three phases i.e., mild, moderate, and severe. The present study
determines that moderate anemia is high in pregnant women as compared to other. The
percentage of mild, moderate, and severe is 22.0%, 55.3%, and 18.2% respectively.
Also, the percentage of anemia among un educated pregnant women is high about 65%.
Moderate anemia is also determined in educated pregnant women. We found that in second and
third trimester of pregnancy, mostly pregnant women become the victim of any type of anemia
because the fetus requirements increase constantly. Mostly organs and body parts are formed in
these months so the requirements of fetus increase.
We also revealed the fundamental causes of anemia during pregnancy. The current
investigation found that a deficiency in iron, folic acid, or vitamin B12 commonly results in
anemia. Megaloblastic anemia is brought on by folic acid and vitamin B12 deficiency, whereas
iron shortage results in iron deficiency anemia. 51.5 % pregnant women take iron supplements
that is mean these are all victims of iron deficiency anemia. And 22% patients take folic acid or
B12 supplements, it shows that they have megaloblastic anemia.
Other factors which are discussed here are hypertension, miscarriage, abnormality in
menstrual cycle etc. These all factors cause anemia in different ways.

39
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43
Questionnaire

A Survey Based Study to Find out the prevalence of Anemia and associated factors in
pregnant women in South Punjab.

We are students of BS Medical Laboratory Technology (8th semester) in Khawaja Fareed

university of engineering and Information Technology, Rahim Yar Khan. We have been assigned
to do a survey on to find out the prevalence of anemia and associated factors in pregnant women
in South Punjab. This survey is purely for educational and awareness purpose. We assure you
that the obtained data will be confidential in every respect. We shall appreciate your response.

The questionnaire consists of three parts. First part was concerned with demographics of the
respondents. The second part consist of survey based upon associated factors of anemia and the

third part differentiate severity of anemia.

Table: 1 Demographics Characteristics

44
 Age

18-22

23-26

27-30

31-34

Table: 2 35………
Survey
Residence
based upon
associated Rural
factors of Urban
anemia
regarding
pregnancy

Month of pregnancy

1-3

4-6

7-9

Questions Yes No Don’t Know value

• Education

• Hb% of the respondent

• House wife

• Doing job

• Do you have any pregnancy before?

45
•Do you have more than four pregnancies?

•Do you eat fresh fruits, vegetables and have milk daily?

• Are you using any sort of iron supplements?

• Are you using any sort of B12 or folic acid supplements?

• Are you suffering from nausea and vomiting?

• Do you have previous history of miscarriage?

• Are you suffering from any hemorrhagic disease?

• Do you take exercise daily?

• Are you suffering from viral infections?

• Are you suffering from stress or hypertension?

• Do you have normal Hb% before pregnancy?

•Is your Hb% low in every pregnancy?

•Do you feel any abnormality during menstrual cycle?

•Do you suffer from lethargy and light headedness During pregnancy?

Table: 3 Severity of anemia

Severity of anemia Hb%

Normal Above 11.5

Mild 10.0-11.5

46
Moderate 8.00-9.9

Severe Below 8.00

47