Magnetic Nanoparticle-Based Biosensing Assay Quantitatively Enhances Acid-Fast Bacilli Count in Paucibacillary Pulmonary Tuberculosis

Abstract

A new method using a magnetic nanoparticle-based colorimetric biosensing assay (NCBA) was compared with sputum smear microscopy (SSM) for the detection of pulmonary tuberculosis (PTB) in sputum samples. Studies were made to compare the NCBA against SSM using sputum samples collected from PTB patients prior to receiving treatment. Experiments were also conducted to determine the appropriate concentration of glycan-functionalized magnetic nanoparticles (GMNP) used in the NCBA and to evaluate the optimal digestion/decontamination solution to increase the extraction, concentration and detection of acid-fast bacilli (AFB). The optimized NCBA consisted of a 1:1 mixture of 0.4% NaOH and 4% N-acetyl-L-cysteine (NALC) to homogenize the sputum sample. Additionally, 10 mg/mL of GMNP was added to isolate and concentrate the AFB. All TB positive sputum samples were identified with an increased AFB count of 47% compared to SSM, demonstrating GMNP’s ability to extract and concentrate AFB. Results showed that NCBA increased AFB count compared to SSM, improving the grade from “1+” (in SSM) to “2+”. Extending the finding to paucibacillary cases, there is the likelihood of a “scant” grade to become “1+”. The assay uses a simple magnet and only costs $0.10/test. NCBA has great potential application in TB control programs.

Nanoparticle-Based Biosensing Assay for Universally Accessible Low-Cost TB Detection with Comparable Sensitivity as Culture

“…The images show that the GMNP aggregates and concentrates the AFB when using NCBA ( Figure 6A); the same sample registered negative (no visible red AFB) when using SSM ( Figure 6B). Our previous study showed that NCBA increased AFB count by 47% compared to SSM [33].…”

“…The images show that the GMNP aggregates and concentrates the AFB when using NCBA ( Figure 6A); the same sample registered negative (no visible red AFB) when using SSM ( Figure 6B). Our previous study showed that NCBA increased AFB count by 47% compared to SSM [33]. Using a medical statistical software (available online: https://www.medcalc.org/calc/diagnostic_test.php, accessed on 11 December 2019), the clinical sensitivity, clinical specificity, positive predictive value, and negative predictive value were calculated and presented in Table 2.…”

“…This growth rate matched the reported doubling rate of Mtb at 15 h [46]. The estimated carrying capacity was set to 10 5 CFU/mL, equivalent to a smear grade of 4+ (many) [33,34] and equivalent to a bacterial load in a patient having been sick for 8 months without receiving treatment. Table 3 column 3 shows the result of the estimated Mtb load based on the logistic growth model.…”

“…Table 3 column 3 shows the result of the estimated Mtb load based on the logistic growth model. The table shows that Mtb count increases from an initial infection at the beginning of the month to 6 CFU/mL at the end of the first month and grows to 9 × 10 4 CFU/mL at the end of the fifth month at which time the infection is detectable by SSM in the sputum sample, equivalent to a smear grade of 1+ (rare) or 2+ (few) [33]. Clinical observations have shown that, although bloody sputum is a sign of TB, smear microscopy does not always detect these cases, especially at early onset of hemoptysis.…”

“…Our previous studies on NCBA for TB detection included quantitative capture efficiency and concentration factor in sputum samples by Gordillo et al [33] and Bhusal et al [34]. In Gordillo's study, we determined that NCBA could concentrate acid-fast bacilli (AFB) Mtb by 47% compared to SSM.…”