Research ArticleDiagnostic Yield of Fluorescence and Ziehl-NeelsenStaining Techniques in the Diagnosis of PulmonaryTuberculosis: A Comparative Study in a District Health Facility

Eben Godsway Dzodanu,1 Justice Afrifa ,1,2

Desmond Omane Acheampong ,3 and Isaac Dadzie 1

1Department of Medical Laboratory Science, University of Cape Coast, Cape Coast, Ghana2Scientific Research Center, �e Second Affiliated Hospital of Harbin Medical University, Harbin, China3Department of Biomedical Sciences, University of Cape Coast, Cape Coast, Ghana

Correspondence should be addressed to Isaac Dadzie; idadzie@ucc.edu.gh

Received 8 February 2019; Accepted 27 March 2019; Published 10 April 2019

Academic Editor: Amit Kaushik

Copyright © 2019 EbenGodswayDzodanu et al.This is an open access article distributed under theCreative Commons AttributionLicense,whichpermits unrestricteduse, distribution, and reproduction in anymedium, provided the original work is properly cited.

Background. Despite the recent advancement in diagnostic methods, the smear microscopy remains the gold standard for thediagnosis of pulmonary tuberculosis in high burden countries like Ghana. Notwithstanding, fluorescence staining techniqueprovides a more efficient option for the detection of Mycobacterium tuberculosis positive smears. This study therefore aimed atassessing the diagnostic performance of fluorescencemicroscopy (FM) andZiehl-Neelsen (ZN) staining techniques in the diagnosisof pulmonary tuberculosis. Methods. A comparative study was carried out on 100 patients who reported at the Out PatientsDepartment (OPD) or the Directly Observed Therapy (DOT) center of the Kade Government Hospital and were suspected ofhaving pulmonary tuberculosis. Two (2) sputum samples each were collected. This included one spot and one morning sample.The smears were prepared and stained with FM and ZN staining techniques. Xpert MTB/RIF assay was also performed. Results.Of the 200 samples analyzed, 71 (35.5%), 46 (23.0%), and 84 (42.0%) were positive for pulmonary tuberculosis when FM, ZN, andXPERT MTB/RIF assays were used, respectively. The mean reading time of FM was three times faster than the ZN technique withvery good acceptance (1.5min: 4.6min).The sensitivity and specificity of fluorescent staining to that of XPERTMTB/RIF assay were84.5% and 100%, respectively, while those of ZN staining were 54.8% and 100%, respectively. Conclusion. For a routine laboratorytest in a resource-limited setting, our study has demonstrated that fluorescence staining technique is a more sensitive test for thediagnosis of pulmonary tuberculosis as compared to the conventional ZN technique.

1. Introduction

Tuberculosis caused by Mycobacterium tuberculosis remainsa major public health problem with approximately one-thirdof the world’s population affected. In 2017, 10 million peoplewere infected with tuberculosis and 1.6 million died from thedisease. Over 95% of tuberculosis deaths occur in low- andmiddle-income countries.[1].

A faster, simpler, more accurate, and less expensive meansof diagnosis of tuberculosis is necessary for the control ofpeople infected with the disease as wells as preventing itsspread in the community [2]. Various investigations canbe used to help in the diagnosis of tuberculosis, and these

include chest radiographs, clinical suspicion, staining foracid-fast bacilli, culture for mycobacteria, and nucleic acidamplification assays. Sputum smear microscopy is the mostpreferred and rapid test that is widely used for the detectionand diagnosis of pulmonary tuberculosis [2, 3]. The bacilliin the sputum can be detected either by ZN or fluorescencestaining techniques. Sputum microscopy is helpful to assessthe response to treatment and to establish a cure or failure atthe end of treatment.

In many developing countries, the diagnosis of tuber-culosis is mostly based on the ZN staining technique [4].The sensitivity of sputum smear microscopy by ZN method,however, is reported to be low and variable, ranging from20%

HindawiTuberculosis Research and TreatmentVolume 2019, Article ID 4091937, 6 pageshttps://doi.org/10.1155/2019/4091937

2 Tuberculosis Research and Treatment

to 80%, often depending on the diligence with which speci-mens are collected, smears are made, and stained smears areexamined [4–7]. This procedure leaves a significant numberof cases undetected, especially if it becomes the only meansof diagnosis. FM was introduced to improve the outcomesof sputum smear microscopy. The sensitivity of conventionalFM provides far better yield and detection of positive smearsthan the ZN and takes less time to perform [8–10]. Thereis however a lingering doubt about the specificity of FM asthere is the possibility of false positives which may be due tothe incorporation of fluorochrome dyes by inorganic objects[11, 12]. Additionally, cost constraint is a limitation of the FM[13] especially, in a low to medium income countries such asGhana.

In Ghana, diagnosis of pulmonary tuberculosis hasinvolved the use of conventional light microscopy to examineZN stained direct smears. The FM is gradually replacingZN stained smear microscopy, while the use of moleculartechniques for TB diagnosis is mainly limited to the teachingand regional hospitals. There is currently no documentedevidence in the country evaluating the application of thetechniques for the diagnosis of pulmonary tuberculosis.Hence, this study compares the diagnostic efficacy of thesethree methods for the detection of pulmonary diagnosis oftuberculosis.

2. Materials and Methods

This prospective study was carried out at Kade Govern-ment Hospital in the Eastern Region by trained biomed-ical scientists. Samples were collected between 08:00 amand 12:00 noon each day. The study population consistedof patients suspected of pulmonary tuberculosis and havereported at the OPD/DOT center of the Kade GovernmentHospital. A total of 100 patients were recruited for thestudy.

2.1. Sample Collection and Preparation. The study subjects(hundred patients) were requested to submit two sputumsamples each in a clean, sterile, leak-proof, wide-mouthcontainers. In total, 200 samples were collected. One samplefrom each patient was taken on the spot and the subjects wereprovided with a second prelabeled container for a morningsample to be taken at home. Preparation of smear for stainingwas done as described elsewhere [14]. For each sample, thesmears were made in duplicate. Positive and negative controlsmears were also prepared.

2.2. Ziehl-Neelsen (ZN) Staining Procedure. The smears werearranged in serial order on staining bridge, with smear sideup and flooded with filtered 0.1% Carbol Fuchsin.The smearswere steamed and allowed to stain for 5 minutes, rinsedwith water, and drained. They were decolorized with 25%sulphuric acid for 5 minutes, rinsed with water, and drained.They were then counterstained with 0.1% methylene bluesolution for 1 minute and rinsed with water. The smear wasallowed to air dry and examined microscopically using theoil immersion (100x) objective.

2.3. Fluorescence Microscopy Staining Procedure. The smearswere flooded with filtered 0.1% auramine for at least 20minutes. They were then rinsed with water and drained.Acid alcohol decolorizing solution (0.5%) was applied on thesmear for 30 to 60 seconds, rinsed with water, and drained.They were then flooded with 0.5% potassium permanganatecounterstain for a maximum of 1 minute and rinsed withwater. The smears were allowed to air dry and examinedmicroscopically using the dry (40x) objective lens of an LEDillumination-based fluorescence microscope (Zeiss primostar ilED).

2.4. Xpert MTB/RIF Assay Procedure. Xpert MTB/ RIF assaywas performed following the protocol of the manufacturer(Cepheid Inc., Sunnyvale, CA, USA). Samples were collectedin containers provided and treated with sample reagent ina proportion of 2:1 and incubated for 15 minutes at roomtemperature. Two milliliters (2ml) of the reagent treatedsample was pipetted into the sample chamber of the Xpertcartridge. The Xpert cartridge was then placed into the Gen-eXpert instrument system and run. Results were generatedafter 90min.

2.5. Ethical Consideration. Study approval was sought fromthe authorities of the Kade Government Hospital. Writteninformed consent was sought from all participants beforerecruitment. Records were kept strictly confidential.

3. Data Analysis

Data were entered in Microsoft, and the statistical analysiswas performed using SPSS software, version 23.0 (IBM Inc.).Data were expressed in percentages for the different variables.Kappa test was used to determine the correlation betweendiagnostic tests and the receiver operating characteristiccurve was used to determine the sensitivity and specificityof tests. P-value <0.05 was considered statistically significant.Xpert MTB/RIF assay was taken as reference when ZN andfluorescence microscopy tests were compared. Samples thatwere positive and negative by GeneXpert were consideredtrue positive and true negative.

4. Results

A total of 200 samples received from 100 individuals werescreened for the presence of acid-fast bacilli (AFB) usingthe ZN and fluorescence microscopy staining techniques. Inthe absence of culture, Xpert MTB/RIF, which amplifies anddetects specific gene target of Mycobacterium tuberculosis,was used as a reference.

Table 1 shows the results of the diagnostic tests usedin the study. Of the 200 samples analyzed, 46 (23.0%),71 (35.5%), and 84 (42.0%) were positive for pulmonarytuberculosis when ZN staining, fluorescence staining, andXpert MTB/RIF assay were used, respectively. Of the 100subjects recruited for the study, FM yielded 37 (37%) whereasZN staining identified 25 (25%) of them to be infected withthe AFB.

Tuberculosis Research and Treatment 3

Table 1: Comparison of diagnostic tests used.

Fluorescent staining Ziehl-Neelsen staining Xpert MTB/RifSpot EM (N=200) Spot EM (N=200) (N=200)

(N=100) (N=100) (N=100) (N=100)Positive 34(34) 37(37) 71 (35.5) 21(21) 25(25) 46 (23.0) 84 (42.0)Negative 66(66) 63(63) 129 (64.5) 79(79) 75(75) 154 (77.0) 116 (58.0)

Sputum GradingScanty 12 (6.0) 2 (1.0)1+ 20 (10.0) 10 (5.0)2+ 21 (10.5) 24 (12.0)3+ 18 (9.0) 10 (5.0)Data is represented in raw figures and percentages. EM: early morning sample.

Table 2: Correlation of fluorescence and ZN staining techniques with Xpert MTB/Rif.

Staining Technique XPERT MTB/Rif Kappa P-value PPV (%) NPV (%)Positive Negative

FluorescencePositive 71 (84.5) 0 (0.0) 0.864 p≤0.001 100 92.1Negative 13 (15.5) 116 (100)ZNPositive 46 (54.8) 0 (0.0) 0.584 p≤0.001 100 77.3Negative 38 (45.2) 116 (100)Data is represented in raw figures and percentages. P-value is statistically significant if p < 0.05 as compared between Xpert MTB/Rif and fluorescencemicroscopy, Xpert MTB/Rif and ZN.

Table 2 compares the results of ZN and fluorescencestaining with the Xpert MTB/RIF test. Of the 84 positivesamples, 38 (45.2%) were missed by ZN staining techniqueand 13 (15.5%) were missed by FM. Both fluorescence andZN staining techniques showed a positive correlation withXpert MTB/RIF diagnostic technique; however, fluorescentstaining had a stronger correlation compared to ZN stain-ing (kappa=0.864, p≤0.001 versus kappa=0.584, p≤0.001). Itcould also be observed that all the samples that were negativeby Xpert MTB/RIF test were also negative by both ZN andfluorescence staining.

Tables 3 and 4 compare the diagnostic performance ofZN to that of FM. Fluorescence staining showed a posi-tive correlation with ZN staining technique (kappa=0.680,p≤0.001). It could be seen that 25 (35.2%) of the samples thatwere positive by fluorescence microscopy were missed by ZNstaining whereas only 1 (0.8%) sample that was positive by theZN staining was negative by FM. The two tests had the samespecificity (100%); however, the sensitivity of fluorescentstaining (84.5%) was higher than that of ZN staining (54.8).The ROC analysis (Figure 1) also proved fluorescent staining(AUC= 0.923) to be a better diagnostic test than ZN staining(AUC=0.774), when the testswere compared to the reference.

Table 5 shows the comparison of the diagnostic yieldof spot and early morning sputum samples as stained byfluorescence and ZN staining techniques. Of the 37 earlymorning samples that tested positive for AFB, 34 (91.8%) oftheir corresponding spot samples also tested positive with3(8.1%) testing negative when fluorescent staining techniquewas used. There was a strong agreement between early

ROC Curve

Source of the CurveFluorescenceZNReference Line

0.0

0.2

0.4

0.6

0.8

1.0

Sens

itivi

ty

0.2 0.4 0.6 0.8 1.00.01 - Specificity

Figure 1: Receiver operator characteristic curve.

morning and spot samples as indicated by the Cohens Kappa(0.935); however, the difference was statistically significant(p<0.05). Also, of the 25 early morning samples that testedpositive for AFB, 21 (84.0%) of their corresponding spot

4 Tuberculosis Research and Treatment

Table 3: Comparison of diagnostic performance of fluorescence staining to ZN staining.

ZN Fluorescence Kappa P-valuePositive Negative

Positive 46 (64.8) 1 (0.8) 0.680 p≤0.001Negative 25 (35.2) 128 (99.2)Data is represented in raw figures and percentages. P-value is statistically significant if p < 0.05 as compared between FM and ZN.

Table 4: Receiver operator characteristic (ROC) curve parameters.

Technique Sensitivity Specificity AUC 95%CI P-valueFluorescence 84.5% 100% 0.923 0.876-0.969 p≤0.001ZN 54.8% 100% 0.774 0.702-0.846 p≤0.001Xpert MTB/Rif used as reference diagnostic test; AUC: area under curve. Data is represented in percentages. P-value is statistically significant if p < 0.05 ascompared between Xpert MTB/Rif and fluorescence microscopy, Xpert MTB/Rif, and Ziehl-Neelsen.

fluorescence Ziehl-Neelsentechnique

0

1

2

3

4

5

time (

min

utes

)

Figure 2: Average time taken to observe fluorescence and ZNstained smears.

samples also tested positive with 4 (16.0%) testing negativewhen ZN staining technique was used.

The mean reading time of fluorescent staining techniquewas three times faster than the ZN technique with very goodacceptance (1.5min: 4.6min) (Figure 2)

5. Discussion

Growth detection of AFB in culture considered the mostsensitive method for diagnosis of TB is not routinely donein our health facilities mainly due to the slow growth ofthe bacteria and the lack of equipment required for the test.Prompt diagnosis of TB, therefore, is achieved by AFB smearmicroscopy, mostly by the ZN technique and recently bythe fluorescence microscopy and the Xpert MTB/RIF in fewfacilities that have them.We compared the results of ZN stainsmear and fluorescence staining with Xpert MTB/RIF test fordetection of AFB in sputum samples.

Results of this study demonstrated that Xpert MTB/RIFdiagnosed approximately 15% more of the 200 samplesscreened than FM and as high as 45% more than ZN. Thesuperior performance of Xpert MTB/RIF over fluorescenceand ZN microscopy in the diagnosis of pulmonary tubercu-losis has been established in many studies [15–18]. Also, FMproduced a higher diagnostic yield compared to that of ZN

staining technique among our study samples. This findingconfirms the previously reported superior performance ofFM over the conventional ZN technique for AFB detection[19–21]. Comparatively, the fluorescence microscopy gener-ated readings at a rate that is approximately three timesfaster than ZN technique, thus helping save approximately2 minutes per slide (66%) which corroborate findings ofa study conducted by Marais, Brittle [22]. Therefore, theintroduction of LED-FMwould be time-saving and allow forquality microscopy.

The study revealed a higher percentage of false negativesfrom ZN staining technique (45.2%) as compared to FM(15.5%) and this is consistent with a study carried out inSinamangal, Nepal [19], which showed that the percentageof false negative by FM staining was only 2.78%, and wasin sharp contrast to that of ZN (40.27%). FM was also ableto detect more paucibacillary cases than ZN. The better casedetection rates of FM over ZN were comparable to reportsfound in several studies [8, 23–25].

A significant benchmark for an alternative diagnosticmethod is its ability to establish a linear relationship withthe gold standard. Interestingly, both FM and ZN tech-niques correlated positively with Xpert MTB/RIF diagnostictechnique. Fluorescent staining, however, showed a strongerlinear relationship as compared to ZN staining (kappa=0.864,p≤0.001 versus kappa=0584, p≤0.001). Elsewhere, Stella etal. observed similar findings when both the FM and ZNtechniques were compared to the PCR method (kappa FMversus PCR=0.60; ZN versus PCR=0.54) [26].

In an ideal situation, a diagnostic technique should be100% specific and 100% sensitive. Our results indicated thatboth the ZN and FM techniques showed similar specificity(100%) when compared to that of Xpert MTB/RIF; however,in relation to XpertMTB/RIF, FM (84.5%)wasmore sensitivethan ZN staining (54.8%). In general, it has been reportedthat, in the diagnosis of pulmonary tuberculosis, the fluo-rescence staining technique provides better sensitivity andspecificity when compared to the ZN [27, 28].

This study showed that, in both FM and ZN stainingtechniques, early morning samples yielded more AFB ascompared to spot samples. Interestingly, sputum samplespositive for smear collected at the spot were also positive for

Tuberculosis Research and Treatment 5

Table 5: Comparison of diagnostic yield of “spot” and “early morning” sputum samples.

Spot EM Kappa P valuePositive Negative

FluorescencePositive 34(91.8) 0(0.0) 0.935 p≤0.001Negative 3(8.1) 63(100.0)ZNPositive 21(84.0) 0(0.0) 0.887 p≤0.001Negative 4(16.0) 75(100.0)Data is represented in raw figures and percentages. P-value is statistically significant if p< 0.05 as compared between “spot” and “early morning (EM)” sputumsamples. EM: early morning.

samples collected early in the morning. This agrees with astudy carried out by Myneedu et al. and which showed thatthe first sputum sample (spot sample) collected immediatelyin the vicinity of the laboratory showed a reduced smearpositivity as compared to morning samples. Similarly, theyalso reported that all AFB positive spot samples were alsopositive for AFB in the early morning smears [29]. To reducediagnostic defaulting, this result calls for a second look atthe standard 2-day protocol of collecting samples on twoconsecutive days for diagnosis of pulmonary tuberculosis.Early morning samples prove to be ideal when onetimesample collection is to be adopted.

In conclusion, our findings show the FM technique to beof a more diagnostic value compared with the ZN technique.It is more sensitive and can detect accurately paucibacillarycases and this has implication on early treatment of pul-monary tuberculosis.

Data Availability

The data used to support the findings of this study areavailable from the corresponding author upon request.

Disclosure

The authors did not receive funding from any funding body.The research was performed as part of the employment of theauthors from the University of Cape Coast.

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this paper.

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