Despite decades of effective treatment being available, tuberculosis (TB) is still in the top 10 causes of death worldwide [1
]. It is caused by the bacteria Mycobacterium tuberculosis
(Mtb), which most often affect the lungs. In 2016, 10.4 million people got sick of TB and 1.7 million died from the disease, of which 95% occurred in low- and medium-income countries [1
]. About one-fourth of the human population is infected with latent TB and 5–15% of this population has a lifetime risk of falling ill with TB [1
In Nepal, TB is the sixth leading cause of death in the country [2
]. It has been estimated that 44,000 people develop active TB every year and 20,500 have infectious pulmonary disease that can spread to others [2
]. In a 2017 national report, TB deaths in Nepal reached 5506, accounting for 3.5% of all deaths and placing Nepal as the 43rd most affected country in the world [3
]. The diagnosis and treatment monitoring of TB patients in Nepal relies on sputum smear microscopy, because it is easy to administer and inexpensive. The country has 596 microscopy centers that carry out sputum microscopy examinations, mostly in government health facilities (487) with the remainder in non-profit organizations and private institutions [3
For decades, many countries have relied on direct (un-concentrated) sputum smear microscopy (SSM) as the primary method for detecting pulmonary TB. SSM is the first microbial analysis both for tuberculosis diagnosis and assessment of patient infectiousness, which is used as a guide to isolation measures and contact investigations [4
]. The method is fast, simple, inexpensive, and specific for Mtb in areas of high prevalence [9
]. SSM is widely applicable in various populations with different socio-economic conditions [9
]. However, it has significant limitations in its performance. The sensitivity is compromised when bacterial load is less than 10,000 organisms/mL sputum sample [9
]. SSM also has a poor track record in extra-pulmonary tuberculosis, pediatric tuberculosis, and in TB patients suffering from the common comorbidity of human immunodeficiency virus (HIV) [9
]. Due to the requirement of serial sputum examinations, some patients do not come back for the repeated sampling or for the exam results, leading to lost opportunity for treatment and follow up. It has been reported that many of the TB diagnoses happen after multiple health care visits and lengthy delays, with an average delay of 28–30 days from the patient’s first contact of a healthcare provider to diagnosis [11
Recently, several methods have been developed for the diagnosis and concentration of TB and multi-drug resistant tuberculosis (MDR-TB), such as Xpert MTB/RIF, TB beads, liquid culture, centrifugation, and filtration [12
]. While these diagnostic methods are more sensitive and/or specific than SSM, they are oftentimes prohibitively expensive and not too accessible for those living in low-resource countries where Mtb has a high prevalence.
The World Health Organization recommended the Xpert MTB/RIF in 2010 to diagnose all persons with signs and symptoms of TB. In 2016, the public sector procured 6.9 million cartridges under concessional pricing, but this is still far less than the number that would be needed to diagnose the more than 10 million people who fall ill with TB each year [11
The difficulty faced by TB care and prevention efforts has been the issue of rapid, reliable, and universally accessible diagnosis. Rapid TB diagnosis is one of the most important steps for instituting control measures. Faster TB diagnosis means earlier treatment and epidemiological control. Without proper diagnosis, disease treatment can become a trial-and-error effort. Unfortunately, accurate and timely diagnosis of TB is still wanting.
The ambitious goal of the global “End TB Strategy” to reduce TB incidence to 90% and reduce TB mortality to 95% by 2035 will not be achieved without new tools to fight TB [17
]. These tools include improved point-of-care diagnostic tests that can be delivered in communities and at the first point-of-contact by patients in the healthcare system [17
]. These tests should be done on an easily accessible sample and results be provided in a timely manner, allowing for a quick turnaround time for treatment in a single clinical encounter, hence avoiding loss of patient follow up [17
One of the most important research developments in modern science is nanotechnology. It allows scientists, engineers, chemists, and physicians to work at the molecular and cellular levels to produce significant advances in the life sciences and healthcare. A new method of detecting TB in pulmonary cases that are based on nanoparticle science is presented in this paper. The objective of this study was to combine principles in nanotechnology, TB biology, glycochemistry, and engineering for the development of a nanoparticle-based colorimetric biosensing assay (NCBA) to quickly and inexpensively detect very low concentrations of acid-fast bacilli (AFB) in sputum samples. The novelty of NCBA includes the utilization of iron oxide nanoparticles with superparamagnetic properties. The use of magnetic nanoparticles (MNPs) offers major advantages due to their unique size and physicochemical properties. The MNP solution is colloidal in nature and it provides stability, which gives rise to both steric and coulombic repulsions. Their nanoscale size results in their higher effective surface areas, lower sedimentation rates, and minimal precipitation due to gravitation forces [18
]. The MNPs are coated with glycan to facilitate attachment on the bacterial cell wall through carbohydrate-binding protein sites, providing specificity to the biosensing mechanism. In this study, NCBA’s detection performance was compared with the widely used SSM method and the WHO-recommended Xpert MTB/RIF system.
2. Materials and Methods
2.1. Chemicals and Reagents
Carbol fuchsin (0.3%, primary stain) was prepared by dissolving 50 g phenol in 100 mL 90% ethanol and then adding 3 g basic fuchsin in the mixture. Distilled water was added to bring the total volume to 1 L. The decolorization solution was 25% sulphuric acid. The counter stain was 0.3% methylene blue. Xpert MTB/RIF reagent from the kit (Cepheid, Sunnyvale, CA, USA) was used to decontaminate and digest the mucoid sputum sample.
Glycan-coated magnetic nanoparticles (GMNPs) were provided by the Alocilja Research Group from Michigan State University (East Lansing, Michigan, USA). GMNPs consisted of iron oxide (III) or magnetite (Fe3O4) core and a glycan (chitosan) shell. Fe3O4 was synthesized using ferric chloride hexahydrate (FeCl3·6H2O) as precursor in a mixture of ethylene glycol (as reducing agent) and sodium acetate (as porogen). Chitosan was polymerized to surface-modify the iron oxide nanoparticles. Glycan coating on MNP allows for the simple and inexpensive capture of Mtb cells through glycan-glycoprotein interaction without the use of expensive antibodies or aptamers. The Mtb cell envelope is rich in complex carbohydrates and glycoproteins that bind with the glycan functionalized nanoparticles. While glycan-glycoprotein interaction is not necessarily specific to pulmonary M. tuberculosis, environmental and non-pathogenic mycobacteria do not usually manifest in pulmonary sputum samples.
Instruments used in the study included Xpert MTB/RIF polymerase chain reaction (PCR) machine (Cepheid, Sunnyvale, CA, USA), bright field microscope (Olympus CX41, Japan Inc.), and a three-dimensional (3-D) printed magnetic rack containing Neodymium magnets.
2.3. Clinical Samples
In this study, leftover pulmonary samples that were collected between January 2018 and April 2018 sent to the Department of Medical Microbiology, Kathmandu University Dhulikhel Hospital, were investigated. These samples were spontaneously produced sputum specimens early in the morning from study patients for three successive days. Sputum of patients clinically suspected of TB having symptoms, such as coughing for about two weeks, fever, weight loss of greater than 3 kg or dyspnea, or having radiographic imaging features of TB, were collected in sterile screw-capped containers prior to treatment. A total of 500 sputum specimens from 500 TB-symptomatic patients were used in the study. There were no samples from patients asymptomatic of TB or unrelated to TB.
2.4. Sputum Sample Processing
Sputum samples were tested for TB using three techniques: conventional sputum smear microscopy (SSM), Xpert MTB/RIF as the gold standard, and the nanoparticle-based colorimetric biosensing assay (NCBA). Sputum samples collected from a patient in the first two days were stored in the refrigerator. Sputum sample obtained on the third day from the same patient was tested for the presence of acid-fast bacilli (AFB) by SSM and the remainder was combined with the earlier two samples amounting to about 4 milliliters (mL). The combined sputum specimen was divided into two portions and subjected to the Xpert MTB/RIF and NCBA technique. A schematic diagram of the sample processing protocol is presented in Figure 1
Sample testing and data collection for Xpert MTB/RIF and SSM were blinded to the investigator, that is, SSM and Xpert testing were done by a lab technologist while the NCBA testing was conducted by the investigator. The three testing techniques are described, as follows:
Sputum Smear Microscopy (SSM): A smear was prepared by the Ziehl–Neelsen (ZN) method of the third (fresh) sputum sample following the standard protocol by the International Union Against Tuberculosis and Lung Disease [19
] within 2 h after receipt at the hospital. Briefly, about 20 µL of fresh sputum sample was placed on a glass slide and heat fixed by a passing flame from a Bunsen burner. The slide was placed on a staining rack and 0.3% carbol fuchsin was poured over the smear. The underside of the slide was gently heated by passing a flame under the rack until fumes appeared. After cooling for about 5 min, the smear was rinsed with distilled water until no color appeared in the effluent. The smear was washed with 25% sulphuric acid several times until the smear appeared light pink in color. The smear was washed with distilled water and then the counter stain (0.3% methylene blue) was added to cover the smear. Distilled water was used to wash off the counter stain and then the smear was air-dried. Once ready, the smear was examined under a light microscope using 100× oil immersion objective to observe the presence of red-colored AFB.
Xpert MTB/RIF (Cepheid, Sunnyvale, CA, USA): Decontamination reagent from the kit was added to the combined sputum sample in a 2:1 (reagent to sample) ratio in a 15-mL falcon tube, which was then manually agitated twice (or quick vortex) during a 15-min incubation period at room temperature. Subsequently, 2 mL of the decontaminated sputum sample were transferred to the test cartridge using a sterile disposable pipette (provided with the kit). The cartridge was loaded into the Xpert MTB/RIF PCR machine and operated for 1 hour and 50 min. At the end of the real-time PCR run, the result was generated [20
Nanoparticle-based Colorimetric Biosensing Assay (NCBA): About 1 mL of the decontaminated sputum sample was added into a 1.5 mL tube containing 100 µL of the nanoparticles. The GMNP and sputum were mixed and allowed to incubate for 5 min at room temperature. The tube was then placed in a magnetic rack to separate the magnetic GMNP-AFB complex and the supernatant was discarded. A smear was prepared by adding 20 µL of the concentrated GMNP-AFB complex on a glass slide following the SSM procedure, as described in Section 1
2.5. Statistical Analysis
Using Xpert MTB/RIF results as the gold standard, sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and accuracy for SSM and NCBA were calculated at 95% confidence interval (CI) using the online MedCalc statistical software (https://www.medcalc.org/calc/diagnostic_test.php
The synthesized GMNPs occur in clusters with several iron oxide nanoparticles being enclosed in the glycan polymer, as shown in Figure 2
. The iron oxide nanoparticles have an average size of 99±58 nm. These GMNPs are superparamagnetic, that is, their magnetization on average is zero, but an external field is able to magnetize the nanoparticles. The prepared GMNP for this study has been stored at room temperature and it has been stable for at least 12 months.
Five hundred sputum samples that were obtained from 500 patients were included in the study. All 500 samples were tested for TB using SSM, Xpert MTB/RIF, and NCBA. For the SSM test, 32 were positive (32+) and 468 were negative (468−); for the Xpert test, 80 were positive (80+) and 420 were negative (420−); and, for the NCBA test, 80 were positive (80+) and 420 were negative (420−). The 32+ SSM samples were all positive in Xpert and NCBA. Of the 468− (negative) SSM samples, 48 were positive in both Xpert and NCBA. Figure 3
shows a Venn diagram of the distribution of the results from the three tests. Table 1
presents the results from SSM test (not shaded) and NCBA test (shaded) using Xpert MTB/RIF as the gold standard in defining the number of true TB cases and non-TB cases.
Statistical analysis of the diagnostic comparison between SSM and NCBA, using Xpert MTB/RIF results as gold standard for true cases, are presented in Table 2
. At 95% confidence interval, the results show that SSM has a sensitivity of only 40% (29%∓52%), while NCBA has a sensitivity matching that of the Xpert system (95%∓100%). Probably because sputum samples were from suspected TB patients, the specificity, positive predictive value (PPV), and negative predictive value (NPV), for SSM and NCBA are very high close to 100%. The accuracy of SSM is 90% (87∓93%), while the accuracy of NCBA is 100% (99%∓100%). Given the sample size and nature of the collected samples, the calculated prevalence for this cohort of patients is 16% (80 out of 500).
The Xpert MTB/RIF system reports bacterial load as very low, low, medium, and high. These categories were used to estimate the bacterial load in SSM and NCBA by matching the corresponding samples with the Xpert system. Table 3
shows a comparison of the detection limit and dynamic range of detection of the two techniques with respect to the Xpert system. Results from NCBA match well with the results from the Xpert MTB/RIF at all levels. On the other hand, SSM could not detect at very low level, could detect only 14% at low level, 48% at medium level, and 79% at high level. A graphical analysis was conducted on the SSM and NCBA results with respect to Xpert MTB/RIF as the gold standard and the results are presented in Figure 4
. Sensitivity of NCBA matches well with the Xpert system at all bacterial loads, while SSM increases linearly with increasing bacterial load (y = 0.27x − 0.33, R2
= 0.97, Figure 4
Left). TB positive samples are normally distributed around the medium level (Figure 4
Right). SSM has 48% detection at the medium level, which is very close to the calculated sensitivity of 40% (29–52%) in Table 2
shows typical NCBA results for TB+ and TB− sputum samples, as viewed through the eyepiece of the bright field microscope. A TB+ sample (Figure 5
A) shows clumped red GMNP-AFB complexes surrounded by dull brown nanoparticles, while a TB- sample (Figure 5
B) shows dispersed brown nanoparticles.
This paper demonstrates the successful validation of the NCBA tests in 500 samples by the Xpert MTB/RIF, with the results showing perfect agreement. With the Xpert system as the gold standard, the sensitivity of NCBA is in accordance with the Xpert system (100%), while SSM’s sensitivity is only 40%. NCBA could detect very low AFB concentration at 102
CFU/mL, two orders of magnitude lower than SSM. This nanotechnology-based detection is rapid (10−20 min), inexpensive ($
0.10/test), simple, and easily scalable. According to Nepal’s Ministry of Health, a TB diagnostic test with 70% sensitivity (and treatment cure of 85%) would save 300,000 lives over the next five years [3
]. This NCBA technique has a high potential to support and transform the TB control program in Nepal and in other high-prevalence low-resource countries. Implementation in rural areas would help to increase case finding and case notification, and would support programs targeted against drug-resistant TB. Nepal has close to 600 microscopy centers that can support the immediate implementation of this technology in the country. Similarly, it is applicable in many of the high TB-burden countries. This technique can be performed in rural communities and at the first point-of-contact by patients in the healthcare system. Results are obtained in less than 30 min, allowing for a quick turnaround time for treatment in a single clinical encounter. Globally, Desikan hypothesized that a universally accessible and rapid detection method with a sensitivity of 85% and specificity of 97% could potentially save 392,000 adjusted lives annually [9
]. This NCBA technology is poised to enhance the “End TB Strategy” towards a TB-free world.