The first generation tuberculosis (TB) laboratory examination method (Charibaldi dan and Harjoko, 2013) occasionally used in developing countries is based on Ziehl–Neelsen (ZN) staining as the gold standard, with 50 to 60% sensitivity (Fend et al., 2006). Nevertheless, in which TB negative samples examined as positive ZN staining could occur infiltration (Gibson et al., 2009). This ZN staining method works microscopically. Research (Kolk et al., 2010) concluded that the ZN staining method still has unsatisfying result; moreover, TB has become a global public health threat and a health emergency in developing countries. Hence, the development of TB laboratory examination method, which is more sensitive, quicker, more applicable, more portable, and cheaper (Bruins et al., 2013; Zhou et al., 2011), is needed.
The TB laboratory examination methods are newer compared to the ZN staining method. The first type of TB laboratory examination method is based on nucleic acid amplification, such as polymerase chain reaction (PCR), strand displacement amplification (SDA), and transcription-mediated amplification (TMA). The second type is based on serology and cytokine released metal rate examination. Both methods are considered to be more advanced. These two methods are commonly available in developed countries, researching continually TB examination methods. In developing countries, examination methods other than ZN staining are considered as complicated and expensive to be applied widely. Hence, a new approach, therefore, is required for the development of the TB laboratory examination methods (Pavlou et al., 2004). There is a TB laboratory examination method that uses thorax X-ray irradiation (also called the generation; Charibaldi dan and Harjoko, 2013). This method has joined in the TB laboratory examination procedure in some developing countries, but this laboratory examination method is not suitable to be implemented in countries with low income yet with high prevalence.
The next generation of the development of the TB laboratory examination method has used an electronic-nose device by detecting a volatile organic compound (VOC). VOC is an organic compound evaporating quickly. A VOC is hypothesized reasonably as containing an active pulmonary TB biomarker that is derived from infected organisms (a metabolite of TB Mycobacterium) or from infected hosts (a result of oxidative stress) (Phillips et al., 2010). The main problem in the identification of VOC is the pattern similarity of the electronic-nose response curve resulting from the low sensitivity of the sensing system selectivity (Polikar et al., 2001).
Previous research studies aimed at the development of the method detecting the presence of Mycobacterium tuberculosis (MTB) in patients’ sputum using an electronic-nose sensing device. These research studies were carried out using various ways to produce different specificities and sensitivities, as presented in Table 1.
Table 1
Previous researches.
| Reference | Sample type | Classification methods | Results |
|---|---|---|---|
| Fend et al. (2006) | Sputum | Back propagation artificial neural network | Sensitivity 89.09% Specificity 91.14% |
| Gibson et al. (2009) | Sputum | Linear discriminant analysis | Average Sensitivity 80% Average Specificity 75% |
| Kolk et al. (2010) | Sputum | Rob electronic-nose: Linear Discriminant Analysis Partial least square discriminant analysis | Sensitivity 57-64% Specificity 61–70% Sensitivity 42–50% Specificity 73–77% |
| Walter electronic-nose: Linear discriminant analysis Partial least square discriminant analysis | Sensitivity 56-66% Specificity 65–68% Sensitivity 37–61% Specificity 56–67% |
The artificial neural network is implemented to support the development of the TB laboratory examination method. An artificial neural network provides a solution with strong robustness, for approximating real values, discrete values, or vector-valued goal function (Wang and Liu, 2014). An artificial neural network is so far known as the most effective way, for certain types of problems, such as learning to explain complex real-world sensor data (Wang and Liu, 2014). Learning vector quantization (LVQ) is a neural network prototype-based learning (Elly et al., 2013). The prototypes are determined in a training process from training dataset and can be interpreted in a straightforward way as they capture essential features of the data in the same space (Elly et al., 2013).
Therefore, it is necessary to elaborate artificial neural network potentials in some research works, especially to differentiate three different odors and to recognize mixed scents (Kusumoputro et al., 1999). The previous analysis (Kusumoputro et al., 1999) was carried out in an attempt to resolve the weaknesses of LVQ artificial neural network by using fuzzy learning vector quantization (FLVQ) artificial neural network. A result of the research showed that the odor recognition probability of FLVQ artificial neural network is quite high to distinguish the scent of oranges, roses, and cananga flowers. The study (Kusumoputro and Jatmiko, 2002) also revealed that the recognition of mixed odors on the electronic sensing system using the algorithm of FLVQ artificial neural network is better than back propagation (BP) artificial neural network and probabilistic neural network (PNN). The research (Kusumoputro et al., 2002) was carried out to observe the performance of FLVQ artificial neural network for recognizing the mixed odors. Then, the recognition rate of FLVQ artificial neural network was compared to the recognition rate of BP artificial neural network. The combined scents tested in the research consisted of two types of mixtures: first, mixture of two odors and, second, the mixture of three scents. According to this research (Kusumoputro et al., 2002), the result of the recognition of the two or even three-odor mixtures obtained using FLVQ artificial neural network is better compared to that obtained using BP artificial neural network and PNN.
Methods
Main points relating to the development of model are described in this section. First, the below equation based on (Dahlan, 2010) calculating the sample size for this research is explained. Second, the developed block model and tasks of each block are elaborated. Third, various processes of the overall development of model are discussed from the beginning to the end. The developed model in this research is presented in Figure 1.

Figure 1
An HGA-FLVQ Model Block Diagram.
In this research, the sample size was calculated based on (Dahlan, 2010) the following equation:
Here, n denotes the research sample size, Sen is the expected sensitivity of the model of which the diagnostic value was tested, d is expected research precision, α is the accepted error rate to determine the deviation standard (Zα), and P is the prevalence of TB disease (the proportion of positive-TB patients among the TB suspects).
The following is the implementation of Equation (1) in this research. The error rate of type 1 (one direction) that is still accepted (α) equals 5%. The deviation standard (Zα) is 1.96. The expected sensitivity is 90%. The expected research precision (d) is 10%. The TB prevalence (P), referring to the proportion of positive-TB patients after an assessment of overall TB suspects in 2015 who received medical treatments from Prof. Dr. Barmawi Hisyam, Sp.P.D.-KP, equals 70%. Hence, the research sample size is as follows:
It is rounded up to 50 TB suspects.
After receiving Ethics Committee Approval from Medical and Health Research Ethics Committee (MHREC), Faculty of Medicine, Universitas Gadjah Mada, with Ref number: KE/FK/71/EC/2016, primary data retrieval at this research could be started in TB laboratory, Faculty of Medicine, Universitas Gadjah Mada.
The e-nose device employed in this research consisted of six sensors, namely, TGS822, TGS813, TGS2611, TGS825, TGS826, and TGS2620. However, primary data (training data and testing data) of this research were only obtained from three sensor response curves. The sensors used in this research were TGS822, TGS813, and TGS2611, as the other sensors were not adequately responsive and were selective to significantly distinguish the characteristics of patients (relative amplitudes) of the positive ZN staining and the negative ZN staining, using direct eye observation. Each period of the electronic-nose response curve of the three sensors was sampled every 5 sec.
There were three categories of samples, namely 49 negative ZN staining subjects’ sputum samples, 49 positive ZN staining patients’ sputum samples, and 50 sputum samples of TB-suspected patients who received medical treatments at Public Health Centers in Yogyakarta and A RESPIRA Pulmonary Hospital of Yogyakarta Province between February 25 and May 8, 2016. An inclusion criterion of negative-TB samples was when a patient was diagnosed as a TB suspect by the doctor, but the patient was declared contrary by laboratory examination using the ZN staining sputum method. Meanwhile, the inclusion criterion of positive-TB samples was when a positively suspected TB patient was declared positive-TB using ZN staining sputum method. Exclusion criteria from positive-TB samples were +HIV subject and positive-TB in extrapulmonary.
Sensing and sampling processes shown at the first block of Figure 1 are elaborated. The sputum samples of this research were obtained three times. First time, on the first day, the suspected patient’s sputum was obtained when the TB-suspected patient visited the Public Health Center for the first time, and then the TB-suspected patient brought a sputum pot containing the sample on the second day, early in the morning. The second sample was the sputum saved at home, soon after the TB-suspected patient woke up in the early morning on the second day. On that day, the TB-suspected patient brought the sample and handed it over in the sputum pot to the medical officer in the Public Health Center. Third, the sputum sample was accepted again in Public Health Center on the second day, when the TB-suspected patient gave the morning sputum sample. In a biosafety cabinet, the three sputum samples were combined to meet the minimum volume requirement so that evaporation and sensing processes could be carried out. The sputum evaporation of a sample was performed by warming it up at the temperature between 38 and 58°C for 60 min in the electronic-nose sample room. The sensors of electronic-nose devise during the process used VOC as the biomarker to detect the presence of MTB. Then, the sensors calculated the rate of VOC and converted the chemistry magnitude to electrical magnitude (analog signal). After that, an interface application in the electronic-nose device transformed the analog signal of its sensor to a digital signal. The digital signal was presented in the form of response curve from each sensor with its maximum length of 20 cycles. Subsequently, each cycle of the response curve from the electronic-nose device was read or sampled every 5 sec to obtain digital data.
At the second block of Figure 1, the processes consist of two types of pre-processing, namely, noise reduction and feature extraction. Considering that the response curve of the electronic-nose came out from the first block still contained noises, a noise reduction method became essential to apply. The noises could be reduced using a filter as can be seen in Equation (4), and its results were saved in a memory.
Generally, a finite impulse response (FIR) filter as in Smith (1999) for the case of N-Points is shown in the following equation:
Equation (2) can be written as in Kusumoputro et al. (1999) as:
and as in Smith (1999), the case of three-point occurs when M = 2 and b0 = b1 = b2 = 1/3 so that Equation (2) would be the Moving Average Filter Equation used in this research:The second process of the second block (pre-processing) in Figure 1 is feature extraction (the feature of positive-TB and feature of negative-TB) from each curve of the electronic-nose response. The feature extraction could be done after the noises significantly decrease. If marker events in this research could be recognized in each curve of the electronic-nose response, the feature extraction in this research (as pre-processing) could be performed through searching peaks and troughs as landmarks or features (James, 2007). These searching cycles were used to obtain relative amplitudes (Ar) from each cycle in the electronic-nose response curve. Ar became the features and primary data in this research. Response curves of the three sensors of electronic nose were not involved in the processes of feature extraction as an observation unit, because each sensor had the unique response when sensing was performed so that the calculation of Ar was performed for each response curve individually. The response signal of each sensor illustrated amplitude in time function forming a pattern (Kusumoputro et al., 2002). In this research, features of the sensor response signal were extracted using the following equation, as shown in a study Hardoyono et al. (2015) and Figure 2:
Here, A0 is the sensor response calculated by the time of ‘odorant IN’ and Amax is the sensor response calculated by the time of ‘odorant OUT.’
Figure 2
A Cycle of Gas Sensor (TGS) Response in an E-nose Device.
At the third block, there are three processes in the hybrid genetic algorithm-fuzzy learning vector quantization (HGA-FLVQ) model as displayed in Figure 1. In the first process, HGA read and normalized data from the pre-processing result (all of the training data) of each sensor’s output in the pre-processing block.
The second process was the process of assisting initialization of FLVQ artificial neural network so that FLVQ could have an optimal initial cluster center. This process executed HGA operators in the HGA-FLVQ model. Then, the HGA method took the first two Ars of each class (positive-TB and negative-TB) from the training data to become two parents. After that the two parents were processed by simulated-annealing recombination (SAR) function and simulated-annealing mutation (SAM) function in HGA (Adler, 1993). A crossover operator in SAR function and a mutation operator in SAM function were implemented repeatedly. Every time HGA produced two offsprings, the values of fitness from these offsprings were also calculated to determine whether those two offsprings were accepted in the new generation population. If the offspring fitness value was better than the parent fitness value, then that offspring was used to replace its parent. However, if not, then it was ignored, and the Ar cluster center of the final generation of this HGA was used for initialization of FLVQ artificial neural network.
The third process of HGA-FLVQ model was model training performed by FLVQ artificial neural network. In this research, this model training was carried out repeatedly using the sputum samples of a group of 98 patients consisting of 49 positive ZN staining patients, 40 negative ZN staining patients, and 9 healthy volunteers who had never been diagnosed with TB by a doctor. The training process using the FLVQ method can be elaborated as follows. Initialization of the initial cluster center was performed by taking HGA output of each sensor. Then, some variables were calculated repeatedly until the desired minimum error was obtained. Those variables were the weighting exponent (also referred to as fuzziness parameter), learning-rate factor (alpha), learning-rate, new cluster center, and error. Afterward, the last cluster center of each sensor data of each class was saved in a memory as a result of training.
This section also elaborates the result of HGA-FLVQ model training as displayed in Table 2. The process of this HGA-FLVQ model training from TGS813, TGS822, and TGS2611 sensors lasted for 5 to 8 iterations since the error decreased below 0.0001. The error reduction process occurred on each iteration as shown in Figures 3,4, 5.
Table 2
Results of HGA-FLVQ model training.
| TGS813 Sensor data | ||||||
| Iteration | Fuzziness parameter | Learning rate | Cluster center | Error | ||
| 1 | 1.100590 | 0.000987 | 0.001400 | 0.108494 | 0.024354 | 0.0014483775 |
| 2 | 1.101180 | 0.001541 | 0.000924 | 0.140709 | 0.033404 | 0.00111976 |
| 3 | 1.101770 | 0.002089 | 0.000798 | 0.163612 | 0.039210 | 5.5822276E-4 |
| 4 | 1.102360 | 0.002508 | 0.000750 | 0.177564 | 0.042487 | 2.0541892E-4 |
| 5 | 1.102950 | 0.002808 | 0.000727 | 0.186139 | 0.044436 | 7.73241E-5 |
| TGS822 Sensor data | ||||||
| 1 | 1.100590 | 0.001144 | 0.001169 | 0.093718 | 0.212932 | 0.004191581 |
| 2 | 1.101180 | 0.001008 | 0.001355 | 0.097408 | 0.228077 | 2.4299692E-4 |
| 3 | 1.101770 | 0.000919 | 0.001562 | 0.102777 | 0.238711 | 1.41901E-4 |
| 4 | 1.102360 | 0.000857 | 0.001782 | 0.107080 | 0.248823 | 1.207616E-4 |
| 5 | 1.102950 | 0.000807 | 0.002042 | 0.110949 | 0.259704 | 1.3338469E-4 |
| 6 | 1.103540 | 0.000769 | 0.002332 | 0.114258 | 0.270786 | 1.3375138E-4 |
| 7 | 1.104130 | 0.000742 | 0.002620 | 0.116887 | 0.280968 | 1.105932E-4 |
| 8 | 1.104720 | 0.000723 | 0.002887 | 0.118887 | 0.289624 | 7.890986E-5 |
| TGS2611 Sensor data | ||||||
| 1 | 1.100590 | 0.000644 | 0.005906 | 0.031570 | 0.029312 | 0.0014235964 |
| 2 | 1.101180 | 0.001916 | 0.000857 | 0.055587 | 0.020714 | 6.50747E-4 |
| 3 | 1.101770 | 0.002333 | 0.000767 | 0.081313 | 0.015552 | 6.8849424E-4 |
| 4 | 1.102360 | 0.003203 | 0.000704 | 0.096225 | 0.017693 | 2.2693272E-4 |
| 5 | 1.102950 | 0.003725 | 0.000683 | 0.103321 | 0.018730 | 5.1426956E-5 |
| TGS813 Sensor | TGS822 Sensor | TGS2611 Sensor | ||||
| 1st Iteration | ||||||
| The weight of the negative class | −0.051673025 | 0.26946884 | −0.003179715 | |||
| The weight of the positive class | −0.002036426 | 0.1985224 | 0.21423542 | |||
| 2nd Iteration | ||||||
| The weight of the negative class | −0.0356808 | 0.28372976 | −3.8830974E-4 | |||
| The weight of the positive class | 0.028204879 | 0.14191468 | 0.16531087 | |||
| 3rd Iteration | ||||||
| The weight of the negative class | −0.0029952978 | 0.3213401 | 0.00271485 | |||
| The weight of the positive class | 0.052159406 | 0.10385808 | 0.13945574 | |||
| 4th Iteration | ||||||
| The weight of the negative class | 0.03716613 | 0.37145796 | 0.0097912615 | |||
| The weight of the positive class | 0.062625006 | 0.088705875 | 0.1361857 | |||
| 5th Iteration | ||||||
| The weight of the negative class | 0.07502196 | 0.407575 | 0.017326174 | |||
| The weight of the positive class | 0.06537599 | 0.080875896 | 0.12186478 | |||
| 6th Iteration | ||||||
| The weight of the negative class | 0.09876694 | 0.42662817 | 0.02240251 | |||
| The weight of the positive class | 0.06590489 | 0.07604013 | 0.1046309 | |||
| 7th Iteration | ||||||
| The weight of the negative class | 0.11628124 | 0.44089812 | 0.026141549 | |||
| The weight of the positive class | 0.06780671 | 0.074578255 | 0.09289357 | |||
| 8th Iteration | ||||||
| The weight of the negative class | 0.12786691 | 0.44939327 | 0.02856244 | |||
| The weight of the positive class | 0.06953817 | 0.07365509 | 0.08497784 | |||
| 9th Iteration | ||||||
| The weight of the negative class | 0.13485843 | 0.45396066 | 0.029982805 | |||
| The weight of the positive class | 0.07066574 | 0.07268719 | 0.08011716 | |||
| 10th Iteration | ||||||
| The weight of the negative class | 0.13828559 | 0.4564701 | 0.030701837 | |||
| The weight of the positive class | 0.07150341 | 0.072542615 | 0.07794089 | |||
| TGS813 Sensor data | ||||||
| Iteration | Fuzziness parameter | Learning rate | Cluster-Center | Error | ||
| 1 | 1.100590 | 0.001075 | 0.001401 | 0.073105 | 0.072574 | 0.004639 |
| 2 | 1.101180 | 0.001309 | 0.001160 | 0.079481 | 0.068403 | 0.000058 |
| TGS822 Sensor data | ||||||
| 1 | 1.100590 | 0.001113 | 0.001346 | 0.160899 | 0.142446 | 0.004211 |
| 2 | 1.101180 | 0.001361 | 0.001012 | 0.218137 | 0.103790 | 0.004771 |
| 3 | 1.101770 | 0.001516 | 0.000935 | 0.236398 | 0.101749 | 0.000338 |
| 4 | 1.102360 | 0.001731 | 0.000869 | 0.246578 | 0.106190 | 0.000123 |
| 5 | 1.102950 | 0.001982 | 0.000817 | 0.257252 | 0.110136 | 0.000129 |
| 6 | 1.103540 | 0.002266 | 0.000776 | 0.268349 | 0.113576 | 0.000135 |
| 7 | 1.104130 | 0.002558 | 0.000747 | 0.278854 | 0.116367 | 0.000118 |
| 8 | 1.104720 | 0.002829 | 0.000726 | 0.287788 | 0.118478 | 0.000084 |
| TGS2611 Sensor data | ||||||
| 1 | 1.100590 | 0.003575 | 0.000705 | 0.040732 | 0.029254 | 0.001988 |
| 2 | 1.101180 | 0.002175 | 0.000788 | 0.074589 | 0.016031 | 0.001321 |
| 3 | 1.101770 | 0.002993 | 0.000715 | 0.092969 | 0.017238 | 0.000339 |
| 4 | 1.102360 | 0.003588 | 0.000688 | 0.101591 | 0.018471 | 0.000076 |
| Amplitude order | Target class | Class distance 1 | Class distance 2 | Prediction class |
|---|---|---|---|---|
| 1 | 2 | 0.369 | 0.424 | 1 |
| 2 | 2 | 0.357 | 0.392 | 1 |
| 3 | 2 | 0.285 | 0.238 | 2 |
| 4 | 2 | 0.262 | 0.177 | 2 |
| 5 | 2 | 0.337 | 0.318 | 2 |
| 6 | 2 | 0.317 | 0.150 | 2 |
| 7 | 2 | 0.321 | 0.076 | 2 |
| 8 | 2 | 0.310 | 0.087 | 2 |
| 9 | 2 | 0.307 | 0.090 | 2 |
| 10 | 2 | 0.290 | 0.107 | 2 |
| 11 | 2 | 0.286 | 0.111 | 2 |
| 12 | 2 | 0.305 | 0.092 | 2 |
| 13 | 2 | 0.286 | 0.111 | 2 |
| 14 | 2 | 0.284 | 0.113 | 2 |
| 15 | 2 | 0.2880 | 0.109 | 2 |
| 16 | 2 | 0.292 | 0.105 | 2 |
| 17 | 2 | 0.280 | 0.117 | 2 |
| 18 | 2 | 0.288 | 0.111 | 2 |
| 19 | 2 | 0.289 | 0.112 | 2 |
| 20 | 2 | 0.280 | 0.117 | 2 |
| n = 50 | Predicted: No | Predicted: Yes | |
| ZN staining: No | TN = 22 | FP = 4 | 26 |
| ZN staining: Yes | FN = 1 | TP = 23 | 24 |
| 23 | 27 |
| Performance rate | Formula | Result | |
|---|---|---|---|
| Accuracy | (TP+TN)/n | (23+22)/50 × 100% = 90.00% | |
| Error rate | (FP+FN)/n | (4+1)/50 × 100% = 10.00% | |
| Sensitivity (true positive rate) | TP/ZN Staining Yes | 23/24 × 100% = 95.83% | |
| False positive rate | FP/ZN Staining No | 4/26 × 100% = 15.38% | |
| Specificity (true negative rate) | TN/ZN Staining No | 22/26 × 100% = 84.62% | |
| Precision | TP/Predictive Yes | 23/27 × 100% = 85.19% | |
| Prevalence | TP/n | 23/50 ×100% = 46.00% |
| n = 4 | Re-Examination ZN Staining: No | Re-Examination ZN Staining: Yes | |
| ZN Staining: No | TN = 1 | FP = 3 | 4 |
| ZN Staining: Yes | FN = 0 | TP = 0 | 0 |
| 1 | 3 |
| n = 50 | Predicted: No | Predicted: Yes | |
| ZN Staining: No | TN = 22 | FP = 1 | 23 |
| ZN Staining: Yes | FN = 1 | TP = 26 | 27 |
| 23 | 27 |
| n = 50 | Predicted : No | Predicted: Yes | |
| ZN Staining: No | TN=25 | FP=1 | 26 |
| ZN Staining: Yes | FN=3 | TP=21 | 24 |
| 28 | 22 |
| n = 50 | Predicted : No | Predicted: Yes | |
| ZN Staining: No | TN=22 | FP=4 | 26 |
| ZN Staining: Yes | FN=3 | TP=21 | 24 |
| 25 | 25 |
| n = 50 | Predicted : No | Predicted: Yes | |
| ZN Staining: No | TN=25 | FP=1 | 26 |
| ZN Staining: Yes | FN=7 | TP=17 | 24 |
| 32 | 18 |
| n = 50 | Predicted: No | Predicted: Yes | |
| ZN Staining: No | TN=22 | FP=4 | 26 |
| ZN Staining: Yes | FN=7 | TP=17 | 24 |
| 29 | 21 |


