Prevalence and Clinical Implications of Pyrazinamide Resistance in Newly Diagnosed TB Patients in Uganda

Introduction

Tuberculosis (TB) remains the leading cause of morbidity and mortality worldwide as a result of the emergence of drug-resistant Mycobacterium tuberculosis.¹ Drug-resistant tuberculosis is a very serious concern with over 500,000 estimated cases of multi-drug resistant TB/Rifampicin resistance in 2020.² This is a result of a low treatment success rate with only 57% of the patients on conventional treatment regimens achieving success against a set target of 75%.¹ Globally, 10.8 million were diagnosed with tuberculosis in 2023,³ and 10.6 million people were diagnosed with tuberculosis during the year 2022, from the estimates of 10.3 million in 2021 causing approximately 1.3 million deaths recorded in 2024.⁴ The gap between the incident rates and notified cases narrowed to an estimate of 3.1 million in 2022 down from 1.4 million in both years of 2020 and 2021 and then back to the pre-pandemic level of 2019.⁵

Mycobacterium tuberculosis is one of the communicable diseases that are still major ill health causes as well as a leading cause of death globally. During the previous coronavirus pandemic period, TB was ranked as the lead cause of death with a single infectious agent, putting it above Human Immunodeficiency Virus/Acquired Immunodeficiency Syndrome.⁶

Pyrazinamide (PZA) is one of the first-line anti-tuberculosis drugs that perform a critical role in the management of both drug-susceptible and multidrug-resistant tuberculosis, especially in the early stages of infection.⁷ The antibacterial activity of PZA requires an enzyme, known as pyrazinamide, which is a pncA gene encoded found in PZA-susceptible tubercle bacilli. The amidase converts the pyrazinamide to pyrazinoic acid (POA), which has antituberculosis properties. It has also been reported that the pyrazinamidase activity of a given strain of M. tuberculosis correlates with its susceptibility to PZA⁸ and any mutational changes within the pncA gene can lead to pyrazinamide resistance.⁹ However, resistance to pyrazinamide has emerged as a growing global concern, especially in regions with high TB burden and HIV co-infection.¹⁰

Recent studies show that pyrazinamide resistance is increasingly prevalent, with a global rate of approximately 11% to 58% in multi-drug resistant (MDR) TB cases.^11,12 In sub-Saharan Africa, pyrazinamide resistance has been found to vary, but studies suggest a notable increase in areas with high TB and HIV co-infection rates.¹³ In Uganda, while the national TB program has focused on monitoring rifampicin and isoniazid resistance, there are limited data on the prevalence of pyrazinamide resistance, particularly in the context of newly diagnosed TB cases. This gap in data underscores the need for studies focusing on pyrazinamide resistance to better inform treatment strategies.

Pyrazinamide resistance is often linked to the pncA gene, which encodes for pyrazinamidase enzyme.¹⁴ There has been a high occurrence of pyrazinamide resistance among Mycobacterium tuberculosis patients on both standard and new drug regimens, which has indicated a higher demand for routine testing of pyrazinamide resistance through detection of the pncA gene.¹⁵

In Uganda, where HIV prevalence is high and TB is a significant public health concern, understanding the prevalence of pyrazinamide resistance and the role of pncA gene mutations is vital. HIV progression and treatment of TB are well documented, with HIV-positive patients being more susceptible to developing resistant strains due to their immunocompromised status. However, the interplay between pyrazinamide resistance and HIV co-infection in Uganda remains poorly understood.

Methods

Study Design and Setting

This study is a cross‐sectional and laboratory-based study that was conducted at the Department of Microbiology, Bombo General Military Hospital laboratory where sputum positive Mycobacterium tuberculosis samples were collected by simple random sampling technique as well as following the Clinical Laboratory Standard Institute guidelines, 2022 for sputum collection.

The study population comprised newly diagnosed sputum-positive clients confirmed with GeneXpert machine from patients attending the Outpatient department of Bombo General Military Hospital as the entry point into the hospital with the ages between 14 and 80 years.

Sample Size

The formulae of Kish and Leslie (1965) estimated the sample size using the prevalence of 12.8%.¹⁶ The sample size estimation was 166 positive sputum Mycobacterium tuberculosis patients confirmed using the GeneXpert machine.

Laboratory Procedures

Sample Processing

The sample processing was done under biosafety cabinet level 3 for the containment of microorganisms. The GeneXpert assay was employed in this study. This was done by transferring at least 1 mL of the suspended sputum into a conical, screw-capped tube having the Cepheid GeneXpert MTB/RIF Assay for Mycobacterium tuberculosis detection and rifampicin resistance identification. The GeneXpert MTB/RIF test was done for both MTB detection and Rifampicin resistance identification among collected samples before proceeding to molecular techniques.

pncA Gene Amplification

DNA extraction from sputum was done using Cetyltrimethylammonium bromide (CTAB).

The primers used included; forward primer pncA-F (50-GGCCCGATGAAGGTGTCGTA) and reverse primer pncA-R (50-CGGACGGATTTGTCGCTCACTAC). The pncA primers were designed according to the Mycobacterium tuberculosis reference sequence (GenBank accession number AL123456.3).

The PCR master mix was prepared as follows: 2.5µL 10x buffer, 0.5 µL dNTPs, 0.5 Taq polymerase, New England Bio-labs, 0.5µL forward (10µM), 0.5µL reverse (10µM), 3.0µL DNA template and 17.5µL RNAase-Free-H₂O making up to 25.0µL final reaction volume.

PCR Amplification/Cycling

The PCR amplification was carried out in a conventional PCR Thermocycler (CLASSIC K960 Thermal Cycler), a pre-denaturation step at 98°C for 10 min, denaturation at 98°C for 15s, annealing at 61.2°C for 15s, extension at 72°C for 10s, and terminated with final extension at 72°C for 5 min for 40 cycles.¹⁷ Electrophoresis was run at 200V and 80mA for 1 hour. Bands were visualized using the Gene-Flash Trans-illuminator.

Data Analysis

The data were analyzed using STATA software version 17. Categorical variables have been expressed as numbers (n) and percentages (%). All tables have been constructed.

Results

Demographics of the Newly Diagnosed Mycobacterium tuberculosis Patients

A total of 166 sputum-positive Mycobacterium tuberculosis samples were analyzed. The majority (53%, n = 88) were adults aged 30–49 years, with a mean patient age of 36 years (range: 14–80 years). Males constituted 55% (n = 91) of the sample population, and 70% (n = 115) of positive sputum samples were HIV-positive.

Prevalence of Pyrazinamide Resistance Among Newly Diagnosed Mycobacterium tuberculosis Patients

The majority 160/166 (96%) of the newly diagnosed Mycobacterium tuberculosis patients showed no detection of the rifampicin resistance while the rest 6/160 (4%) showed indeterminate rifampicin resistance. Further, still, the data analyzed below reveal the pncA status gene shows more than two-thirds 114 (69%) of the patients did not have any mutations and therefore had no resistance to the Mycobacterium tuberculosis treatment while 2 (31%) of the patients showed resistance to the tuberculosis treatment (Table 1).

Table 1 Demographics of Newly Diagnosed Mycobacterium tuberculosis Patients in Bombo General Military Hospital

Pyrazinamide Resistance Detection Among the Demographics of Newly Diagnosed MTB Patients

The newly diagnosed Mycobacteria tuberculosis patients with pyrazinamide resistance were mainly adults aged between 30 and 49 years. Pyrazinamide resistance was majorly detected in those who had a positive HIV status 29 (56%) (Table 2).

Table 2 Shows Pyrazinamide Resistance Detection Across Demographics of New MTB Patients

Prevalence of Pyrazinamide Resistance Among Newly Diagnosed MTB Patients

Majority 160/166 (96%) of the newly diagnosed Mycobacteria tuberculosis patients showed no detection of the rifampicin resistance, while the rest 6/160 (4%) showed indeterminate rifampicin resistance while using the GeneXpert machine. Further still, the pncA status gene showed more than two-thirds 114 (69%) of the patients were of a negative status indicating no resistance to the tuberculosis treatment. However, 52 (31%) of the patients showed resistance to the tuberculosis treatment.

The Proportion of Rifampicin Resistance in Pyrazinamide Resistance Among Newly Diagnosed MTB Patients

It was observed that only one patient (2%) showed co-existence of pyrazinamide resistance and rifampicin resistance. This was a middle-aged female newly diagnosed with pulmonary mycobacterium, with rifampicin resistance, indeterminate results on GeneXpert and positive HIV status.

Discussion

Pyrazinamide is a very important anti-tuberculosis drug that plays a key role in shortening MTB treatment therapeutic duration by killing the non-replicating persistent MTB hence making it a preferred drug in both susceptible and MDR-TB regimens.^8,17–19 Excessive or increased acidic conditions can also lead to unreliable results of PZA susceptibility tests, hence the inaccurate susceptibility results of PZA leading to the improper treatment of TB and spread of PZA resistance.²⁰

This study provides insight into the prevalence of pyrazinamide resistance among newly diagnosed TB patients in Uganda, highlighting key associations with HIV status. This study illustrated that pyrazinamide resistance is much higher among the youth than in the elderly. The above findings are similar to various research worldwide^6,7,21,22 where the majority were within the age brackets of 30–59 years (60.2%) followed by age below 30 years. The age statistical findings in both studies are similar because of the similar population dynamics where the youths were the majority in both research areas as well as the majority that considered participating in the study.

The majority of the positive pncA gene were male 60% (31/52) which was similar to many other studies.^9,17 The higher prevalence among males may reflect the military setting’s demographics, where men are overrepresented. A higher prevalence of the pncA gene 56% (29/52) among the HIV-positive patients was in agreement with similar findings in Uganda.²³ Additionally, the high prevalence among HIV-positive patients highlights the need for integrated TB and HIV management strategies. However, our study showed a positive moderation association between the HIV status of patients and the presence of the PncA gene, which was contrary to many studies. Having one pncA gene and rifampicin resistance may be attributed to the small sample size used in this study research, hence not mimicking the actual data for the large population. Several studies such as in Pakistan,²⁴ and China¹⁷ reported a higher prevalence in the sample population. This discrepancy may be due to differences in sample populations, prior TB treatment exposure, and local epidemiological factors.

The presence of pncA gene mutations as the primary mechanism of pyrazinamide resistance is well documented.⁹ The enzyme pyrazinamidase, encoded by pncA, is responsible for converting pyrazinamide into its active form, pyrazinoic acid (POA). Mutations in this gene disrupt enzymatic activity, leading to drug resistance.⁹ Given the high prevalence of HIV among TB patients in this study (70%), immunosuppression may contribute to increased genetic variability in M. tuberculosis, potentially facilitating the emergence of pncA mutations. Additionally, co-infection with HIV is associated with more rapid disease progression and altered drug pharmacokinetics, which could influence resistance patterns.¹⁰

These results emphasize the need for routine molecular testing for pncA mutations to enhance TB treatment strategies. Currently, pyrazinamide resistance is not considered in the standard TB diagnostic algorithms, yet its detection is crucial for optimizing therapy, particularly in MDR-TB cases.²⁵ Incorporating pncA mutation screening into routine TB diagnostic workflows could help refine treatment regimens and prevent treatment failure. Furthermore, given the observed association between pncA mutations and HIV-positive status, targeted resistance testing in this high-risk group could improve patient outcomes.

The study sample was limited to newly diagnosed MTB patients, excluding those already undergoing treatment or on second-line regimens. This limits the generalizability of findings to the broader TB population. Lack of genotypic data on specific mutations within the pncA gene that could further explain resistance mechanisms. The study was conducted in a single facility (Bombo General Military Hospital), which may not represent the population dynamics and TB resistance patterns in other regions of Uganda.

Future studies should include a larger and more diverse sample size to capture resistance patterns in different TB populations, including patients on second-line treatment. Strengthen surveillance systems for TB drug resistance as well as incorporate advanced genotypic and phenotypic testing to identify specific pncA mutations and their role in pyrazinamide resistance to guide public health policies and optimize resource allocation.

Conclusion

This study reveals important trends regarding drug resistance and its relationship with HIV status. The majority of patients (96%) did not exhibit rifampicin resistance, suggesting that multi-drug-resistant tuberculosis is not widespread among the newly diagnosed cases.

However, despite the low prevalence of pyrazinamide resistance overall, with only 1 (2%) patient showing resistance to both rifampicin and pyrazinamide, there was a notable association between pyrazinamide resistance and HIV-positive status. The majority (56%) of the patients with the pncA gene mutation, which is associated with pyrazinamide resistance, were HIV-positive. This highlights the potential vulnerability of HIV-positive TB patients to multidrug resistance, even when the overall pyrazinamide resistance rate remains low. Furthermore, this research provided insights into pyrazinamide and rifampicin resistance, highlighting implications for diagnostic accuracy, initial drug regimens, and strategies to mitigate the global TB economic burden.