The use of Kudoh method for culture of Mycobacterium tuberculosis and Mycobacterium africanum in The Gambia

Tijan Jobarteh; Jacob Otu; Ensa Gitteh; Francis S Mendy; Tutty Isatou Faal-Jawara; Boatema Ofori-Anyinam; Binta Sarr; Abi Janet Riley; Abigail Ayorinde; Bouke C de Jong; Beate Kampmann; Ousman Secka; Florian Gehre

doi:10.1371/journal.pone.0300042

. 2024 Mar 27;19(3):e0300042. doi: 10.1371/journal.pone.0300042

The use of Kudoh method for culture of Mycobacterium tuberculosis and Mycobacterium africanum in The Gambia

Tijan Jobarteh ^1,^*, Jacob Otu ¹, Ensa Gitteh ¹, Francis S Mendy ¹, Tutty Isatou Faal-Jawara ¹, Boatema Ofori-Anyinam ^1,⁴, Binta Sarr ¹, Abi Janet Riley ¹, Abigail Ayorinde ¹, Bouke C de Jong ², Beate Kampmann ¹, Ousman Secka ^1,^*, Florian Gehre ^1,^2,^3,^*

Editor: Padmapriya P Banada⁵

PMCID: PMC10971581 PMID: 38536821

Abstract

Background

Mycobacterium tuberculosis culturing remains the gold standard for laboratory diagnosis of tuberculosis. Tuberculosis remains a great public health problem in developing countries like The Gambia, as most of the methods currently used for bacterial isolation are either time-consuming or costly.

Objective

To evaluate the Kudoh swab method in a West African setting in Gambia, with a particular focus on the method’s performance when culturing Mycobacterium africanum West Africa 2 (MAF2) isolates.

Method

75 sputum samples were collected in the Greater Banjul Area and decontaminated in parallel with both the standard N-acetyl-L-Cysteine-NaOH (NALC-NaOH) and the Kudoh swab method in the TB diagnostics laboratory in the Medical Research Council Unit The Gambia between 30^th December 2017 and 25^th February 2018. These samples were subsequently cultured on standard Löwenstein-Jensen and Modified Ogawa media respectively and incubated at 37°C for mycobacterial growth. Spoligotyping was done to determine if the decontamination and culture methods compared could equally detect Mycobacterium tuberculosis, Mycobacterium africanum West Africa 1 and Mycobacterium africanum West Africa 2.

Result

Among the 50 smear positives, 35 (70%) were culture-positive with Kudoh and 32 (64%) were culture positive with NALC-NaOH, whilst 7(28%) of the 25 smear negative samples were culture positive with both methods (Table 2). There was no significant difference in recovery between both methods (McNemar’s test, p-value = 0.7003), suggesting that the overall positivity rate between the two methods is comparable. There were no differences in time-to-positivity or contamination rate between the methods. However, Kudoh yielded positive cultures that were negative on LJ and vice versa. All findings were irrespective of mycobacterial lineages.

Conclusion

The Kudoh method has comparable sensitivity to the NALC-NaOH method for detecting Mycobacterium tuberculosis complex isolates. It is easy to perform and could be an add on option for mycobacterial culture in the field in The Gambia, since it requires less biosafety equipment.

Introduction

Tuberculosis (TB) is an infectious disease caused by bacteria of the Mycobacterium tuberculosis complex (MTBc). Besides M. tuberculosis (MTB), TB in West Africa is also caused by two geographically restricted, endemic mycobacterial MTBc members, Mycobacterium africanum West Africa 1 (MAF1) and M. africanum West Africa 2 (MAF2) [1, 2]. The presence of these two lineages has implications for diagnostic assays and laboratory algorithms [1–3] as most commercially available kits were validated based on global mycobacterial strain collections lacking these two West African lineages. For instance (and in contrast to MTB lineages) strains of MAF2 have a non-functional pyruvate kinase and cannot metabolise glycerol as a sole carbon source [4]. Therefore, growth media in West Africa needs to be supplemented with pyruvate.

Smear microscopic examination is quick to perform and almost universally available, however there is a lack of sensitivity with sputum smear microscopy [5], and it cannot distinguish between live and dead bacilli in treatment monitoring. While molecular detection such as with the GeneXpert approaches the sensitivity of culture, mycobacterial culture isolation remains an important diagnostic test given the improved sensitivity [6], especially for patients with presumptive extrapulmonary TB, and for treatment monitoring of MDR-TB patients. One shortcoming of these culture methods is that they are prone to contamination. Therefore, collected sputa need to undergo thorough decontamination to eliminate other fast-growing contaminating bacteria. Globally, different mycobacterial culture methods are used with Petroff’s method (adding 4% NaOH to the sputum) being one of the most commonly used procedures for mycobacterial culture [7].

In The Gambia, a modified version of the Petroff method (in which a mucolytic agent N-acetyl-L-Cysteine is added to NaOH) had been used as the standard decontamination procedure for sputum samples. This method is cumbersome, and it requires at least 45 minutes of manipulation, including use of a cooled centrifuge, by technically skilled personnel before the sample can be inoculated on LJ slopes. Furthermore, the Centers for Disease Control has recommended biosafety level 3 laboratories for this type of operations [5]. This is one of the important reasons why culturing of sputum samples, especially in rural areas with poorly equipped laboratories, is only available at the National level in The Gambia and most other developing countries.

Therefore, Kudoh and Kudoh [8] described a simplified swab method for decontamination and culturing of mycobacteria that takes only 4–5 minutes per sample. This method does not need expensive equipment such as centrifuges and biosafety cabinets, nor high level technical skills, as a swab is simply dipped into the sputum, directly and aseptically transferred into an NaOH containing tube and then (without centrifugation) inoculated on modified Ogawa medium slopes. We aimed to evaluate the Kudoh swab method in a West African setting in The Gambia, with a particular focus on the method’s performance when culturing MAF2 isolates.

Materials and methods

Ethical consideration

The study was conducted within the framework of TB Sequel study (SCC 1523v1.1.) in the Greater Banjul area of The Gambia. TB Sequel is a multi-country study, in which Gambia is a study site. Within the established observatory TB cohort, TB Sequel investigates the clinical, microbiological, immunological, and socio-economic risk factors predicting the outcome of pulmonary TB. Participants were recruited at both healthcare facilities and the Medical Research Council (MRC) unit The Gambia and sputum samples were collected from participants [9]. Ethical approval was granted by The Gambia Government/MRC joint Ethics Committee. Study participants who could not read or write in English provided oral consent whilst those that can read and write in English provided written inform consent in accordance with the Declaration of Helsinki and were anonymized.

Selection of sputum samples

A total of 75 sputa were examined between 30^th December 2017 and 25^th February 2018 in the TB diagnostics laboratory in the Medical Research Council Unit The Gambia. The TB diagnostics laboratory is a ISO15189 accredited by KENAS. Only sputum samples with a volume more than 2 ml were included in the study. Of the 75 sputa, 67% (50/75) were smear-positive. The grading of positivity was 22 (29%), 11 (15%) and 17(23%) for 1+, 2+ and 3+ respectively.1+ is when sputum contain 10–99 AFB in 100 fields, 2+ for 1–10 AFB per field (check 50 fields), and 3+ for more than 10 AFB per field (check 20 fields), respectively, using WHO standards [10]. The samples were collected from the Greater Banjul area of The Gambia. Samples were processed by a qualified laboratory Technician with a BSc in Biomedical science and more than five years’ experience in a biosafety cabinet for Auramine O staining and both NALC-NaOH and Kudoh methods. The technician was blinded from patients’ information like names, place of residence, etc.

Auramine smear microscopy

Direct smear microscopy slides were prepared by taking a purulent portion of the sputum. The slides were heat fixed on a heat block, stained with 0.1% Auramine O and examined with a fluorescent microscope as previously described [11].

NALC-NaOH method and inoculation on Löwenstein-Jensen slopes

Briefly, an equal volume of NALC-NaOH solution (4%) was added to the specimen, vortexed until liquefied (usually 5 to 20 seconds) and incubated for 15 minutes at room temperature. Phosphate buffer (pH 6.8) was added to the 50-ml level and the sample was centrifuged using a refrigerated centrifuge at 3000 X g for 20 minutes after which the supernatant was discarded. The pellet was re-suspended in 2ml sterile phosphate buffer and 250 μl were inoculated onto each of the two LJ slopes (LJ with glycerol and LJ with pyruvate). Cultures were kept for up to 8 weeks until growth was observed at 37°C.

Kudoh method and inoculation on modified Ogawa medium

The Kudoh swab method was performed as reported by Kudoh and Kudoh (8). Briefly, sputum was gathered onto a sterile cotton swab, and the swab was immersed in a sterile 4% Sodium Hydroxide solution in a falcon tube for 2 minutes. The swab was removed from the falcon tube after 2 minutes and directly inoculated onto two Modified Ogawa slopes (Ogawa with glycerol and Ogawa with pyruvate) by smearing and squeezing the swab over the surface of the media [8]. Cultures were kept for up to 8 weeks until growth was observed at 37°C.

Evaluation of LJ and Ogawa slopes

Cultures (LJ and Ogawa) were read once a week for any growth. Colonies morphologically similar to M. tuberculosis as reported in [12] were identified and slides for microscopy were prepared from colonies growing on the culture media for confirmation of AFB growth. A culture was recorded as contaminated if the slant was covered by contaminating organisms at the end of 8 weeks, and no acid-fast organisms had been identified on the slant.

Spoligotyping of isolates

A loopful of grown colonies was collected, re-suspended in 100μl of Tris-EDTA. The bacteria were centrifuged at 10,000 rpm for 15 minutes, after which the supernatant was discarded. The pellet was re-suspended in 100μl of Tris-EDTA by vortexing briefly and the tubes were placed in a heat block at 99°C for 20 minutes, which lyses the mycobacterial cells to release the DNA in solution. In addition, the tubes were sonicated in a water bath at 100°C for 15 minutes and spun down for 5 minutes at 14,000 rpm. The supernatant (which contains the DNA) was stored at -70°C until ready for use.

Spoligotyping, which involves using multiple synthetic spacer oligonucleotides that are covalently bound to a membrane to obtain different hybridization patterns of the amplified DNA using the direct repeats as a target for invitro DNA amplification, was carried out for species identification from positive AFB growth as described in [13]. For identification of lineages the binary spoligotype patterns were uploaded onto the TB Lineage database.

Data analysis

McNemar’s test was used to evaluate the differences between the results of cultures processed with NALC-NaOH and Kudoh methods.

Results

For a detailed overview of all patients’ characteristics and of patients which tested positive for either of the tests please see Table 1 and S1 Table. Among the 50 smear positives, 35 (70%) were culture-positive with Kudoh and 32 (64%) were culture positive with NALC-NaOH, whilst 7(28%) of the 25 smear negative samples were culture positive with both methods (Table 2). There was no significant difference in recovery between both methods (McNemar’s test, p-value = 0.7003), suggesting that the overall positivity rate between the two methods is comparable. Table 2 also shows that there is no difference in recovery between both methods in terms of new cases and follow-up samples. Only two smear positive samples were contaminated in both methods.

Table 1. Patients characteristics of the study population and patients testing positive with either of the methods.

Samples were collected in the Greater Banjul Area / The Gambia between 30^th December 2017 and 25^th February 2018.

Characteristics	Total study population	Positive with Kudoh Method	Positive with NaLC-NaOH Method
Age,Years
15–24	18	13	14
25–44	31	17	16
45–64	18	10	8
≥65	3	2	1
Unknown	5	-	-
Sex
Male	52	31	30
Female	23	11	9
Case
Follow-ups (on treatment)	17	11	9
New Cases	57	30	29
Unknown	1	1	1
Smear Grades
Positive (1+)	22	15	14
Positive (2+)	11	8	6
Positive (3+)	17	12	12
Negative (NS)	25	7	7
Total	75	42	39

Open in a new tab

Table 2. Comparison of the two culture methods stratified by patient status.

Samples were collected in the Greater Banjul Area / The Gambia between 30^th December 2017 and 25^th February 2018.

Culture Methods
	New Case		Follow-Up		Unknown
Culture Outcome	Kudoh	NALC-NaOH	Kudoh	NALC-NaOH	Kudoh	NALC-NaOH
S⁺C⁺	25	23	9	8	1	1
S⁺C^{- (}*⁾	8	10	5	6	-	-
S^-C⁺	5	6	2	1	-	-
S^-C^-	18	17	-	1	-	-
S⁺C^cdx(*⁾	1	1	1	1	-	-

Open in a new tab

Abbreviations: S⁺ = smear positive; S^- = smear negative; C⁺ = culture positive; C^- = culture negative; C^cdx = culture contaminated.

There was also no significant difference in time to detection between the two methods (Fig 1), as the average time to positivity for the Kudoh method was 37 days (95%CI 33.1–41.7) compared to 38 days (95%CI 33.9–42.1) for the NALC-NaOH method.

However, we observed qualitative differences between the methods. Although twenty-seven (36%) samples were culture positive with both methods, fifteen (20%) samples were culture positive with Kudoh method only (and negative with NALC-NaOH method) whilst twelve (16%) were exclusively culture positive with NALC-NaOH method only (Table 3).

Table 3. Mycobacterium tuberculosis complex detection with different methods.

Spoligotype Family	Culture methods			Total
Spoligotype Family	Kudoh only	NALC-NaOH only	Both Kudoh & NALC-NaOH	Total
MAF 1	-	-	1	1
MAF 2	4	2	8	14
M. tuberculosis	11	10	18	39
Total	15	12	27	54

Open in a new tab

To understand whether the observed qualitative performance differences could be due to culture bias of one of the two methods towards certain mycobacterial lineages (such as MAF2) we spoligotyped all mycobacterial isolates. All isolates were identified as MTB, MAF1 and MAF2; only one MAF1 was identified by both methods compared to eight MAF2 identified in both methods, four MAF2 was identify by Kudoh only and two MAF2 was by NALC-NaOH only (Table 3). After stratifying recovery rates of each culture method by mycobacterial lineage (MAF vs. MTB), no differences were observed (p = 0.8124). Furthermore, comparing ancient (MAF, Indo-Oceanic) lineages with modern MTB lineages, no difference was observed (p = 0.6537) (Table 4).

Table 4. Mycobacterium tuberculosis complex lineages and family detection with different methods.

	Kudoh	NALC-NaOH	p-value (Fisher’s exact test)
Lineage
Ancestral	27	23	0.6537
Modern	15	16	0.6537
Family
MAF	13	11	0.8124
MTB	29	28	0.8124
Total	42	39

Open in a new tab

Discussion

We confirmed that for the diagnosis of pulmonary tuberculosis, sputum sample culture is significantly more sensitive than the direct smear.

Sputa processed with both methods in the present study were cultured on two slopes of LJ and Modified Ogawa media. Sodium pyruvate was included in both LJ and Modified Ogawa media to detect MAF based on reports that glycerol favours the growth of MTB whilst pyruvate is usually required by MAF [14].

There was no difference between both methods in mycobacterial recovery rates and contamination levels in the present study. In the present study, 42 (56%) samples were culture positive with Kudoh and 39 (52%) were culture positive with NALC-NaOH. Also, only two samples were contaminated in both methods in the present study. Findings from a study conducted by Rivas et al., [15] in Uruguay in 2010 on Kudoh and NALC-NaOH culture methods also reported no difference regarding contamination and mycobacterial growth. Several researchers in Asian countries, including Japan, have revealed good results with the Kudoh swab method and Ogawa media [15]. Similar performances were reported when working under field conditions when the standard Petroff decontamination procedure was compared with Kudoh swab method in two Caracas hospitals, José Gregorio Hernández and José Maria Vargas in Venezuela in 2009 [16]. A recent study in Mozambique found comparable results to ours, such as exclusive detection of isolates by either method, yet at a lower magnitude, as well as a contamination rate for Ogawa method of around 4% [17].

In the present study, there was also no difference in time to detection between the two culture methods. These have also been proven in a previous study by Jaspe et al., [16] in Venezuela where Kudoh and Petroff methods were compared. The authors reported no difference between the two methods in the time to detection of bacterial growth.

There was, however, a striking difference in the recovery of mycobacteria. Surprisingly, a significant number of bacteria could only be exclusively detected with either of the two methods, yet we did not see any culture bias towards a certain lineage for any of the two methods. Further studies would be needed to investigate the difference in qualitative performance in our setting and it might be advisable to include an Ogawa slope in addition to the conventional LJ slope in the routine diagnostic algorithm to increase the overall TB culture positivity rate (if solid culture is used for primary isolation of MTBc).

The Kudoh method using the Ogawa-modified medium does not require expensive equipment such as refrigerated aerosol safe centrifuges; it has a shorter turnaround time compared to standard NALC-NaOH method and relies on cheap, stable and readily available reagents [18]. Reduced equipment and fewer biosafety requirements for laboratory personnel are clear advantages of the Kudoh method and would make the method applicable for field work in The Gambia. Moreover, the Kudoh method enhances rapid culturing of sputa at field sites as it eliminates the time-consuming centrifuge steps in laboratories, thus also reducing the work load [16]. By using the Kudoh method, clinical isolates could now be recovered in areas of The Gambia where conventional NALC/LJ-based culturing was impossible to date [16]. Instead of transporting batches of sputa (in transport culture media after days of storage in the fridge) from remote areas to centralized facilities for culturing, the Kudoh-based field culturing could significantly increase recovery rates, with similar contamination rate and time to positivity, even if the slants are later sent to a central facility for incubation at 37°C [18]. This method would allow diagnosis of patients in distant areas, with no current access to TB culture capacity. Although we conducted Kudoh in combination with spoligotyping to differentiate between MAF and MTB in the present study, genotyping will not be necessary for clinical management of patients in peripheral health centres, where the Kudoh method could be implemented in the future.

Limitations

The present study also has some limitations. Due to its small sample size, larger, prospective studies should be done in the future to confirm our results, ideally also in other West African settings where MAF is endemic. We also did not document whether the respective overall positive cultures grew on pyruvate- or glycerol-supplemented slants, yet this should be done in future studies to better understand the recovery of MAF in the Kudoh method. Finally, we did not perform spoligotype analysis on sputum samples that remained culture negative in both approaches, to identify an on overall association of culture positivity and mycobacterial lineage. Moreover, we did not test for a delay in incubation at 37°C, such as when Kudoh based inoculation would be conducted in the field and the slopes would remain at room temperature for several days before incubation.

Conclusion

In conclusion, implementing the Kudoh method in African settings could have two benefits. First, with its acceptable sensitivity, specificity and contamination rates, Kudoh is an efficient way to culture Gambian isolates in the field, especially in regions where the NALC/LJ culture method cannot be currently done due to lack of adequate laboratory infrastructure. Secondly, we also found that some isolates were exclusively only detected with the Kudoh method, and not with NALC/LJ. Therefore, in laboratories where NALC/LJ is routinely done already, the Kudoh method could be used as an “add on” culture method to increase overall culture positivity rates by almost 20%. The routine use of the MGIT and GeneXpert system, a quick, automated method, in conjunction with the culture media (LJ and Ogawa methods) would effectively increase the diagnosis rate to control tuberculosis outbreaks. Furthermore, for the diagnosis of tuberculosis and cases involving RIF resistance, GeneXpert may be helpful. Early diagnosis of pulmonary tuberculosis is crucial for containing the spread of tuberculosis.

Supporting information

S1 Table

(XLSX)

pone.0300042.s001.xlsx^{(22KB, xlsx)}

Acknowledgments

The authors thank all colleagues and study participants for their contributions.

Data Availability

All relevant data are within the manuscript and its Supporting Information files.

Funding Statement

The author(s) received no specific funding for this work.

References

1.de Jong BC, Antonio M, Gagneux S. Mycobacterium africanum—Review of an Important Cause of Human Tuberculosis in West Africa. PLOS Neglected Tropical Diseases. 2010;4(9):e744. doi: 10.1371/journal.pntd.0000744 [DOI] [PMC free article] [PubMed] [Google Scholar]
2.Gehre F, Kumar S, Kendall L, Ejo M, Secka O, Ofori-Anyinam B, et al. A Mycobacterial Perspective on Tuberculosis in West Africa: Significant Geographical Variation of M. africanum and Other M. tuberculosis Complex Lineages. PLOS Neglected Tropical Diseases. 2016;10(3):e0004408. doi: 10.1371/journal.pntd.0004408 [DOI] [PMC free article] [PubMed] [Google Scholar]
3.Ofori-Anyinam B, Kanuteh F, Agbla SC, Adetifa I, Okoi C, Dolganov G, et al. Impact of the Mycobaterium africanum West Africa 2 Lineage on TB Diagnostics in West Africa: Decreased Sensitivity of Rapid Identification Tests in The Gambia. PLOS Neglected Tropical Diseases. 2016;10(7):e0004801. doi: 10.1371/journal.pntd.0004801 [DOI] [PMC free article] [PubMed] [Google Scholar]
4.Keating LA, Wheeler PR, Mansoor H, Inwald JK, Dale J, Hewinson RG, et al. The pyruvate requirement of some members of the Mycobacterium tuberculosis complex is due to an inactive pyruvate kinase: implications for in vivo growth. Molecular Microbiology. 2005;56(1):163–74. doi: 10.1111/j.1365-2958.2005.04524.x [DOI] [PubMed] [Google Scholar]
5.Andrei Tudor C, Simona P., Adina Catinca, G., Florin R., Berlea M., Sabina Ioana C., et al. Alternative technique for culturing sputum for mycobacteria isolation: Feasibility, performance and effect on laboratory quality assessment—A technical note. African Journal of Microbiology Research. 2015;9(19):1294–8. [Google Scholar]
6.Essa SA, Abdel-Samea SA-R, Ismaeil YM, Mohammad AA. Comparative study between using Lowenstein Jensen and Bio-FM media in identification of Mycobacterium tuberculosis. Egyptian Journal of Chest Diseases and Tuberculosis. 2013;62(2):249–55. [Google Scholar]
7.Reider HL CM, Myking H, Urbanczik R, Laszlo A, Kim SJ, Van Deun A et al. The Public Health Service National Tuberculosis Reference Laboratory and the National Laboratory Network. 1998:1–33. [Google Scholar]
8.Kudoh S, Kudoh T. A simple technique for culturing tubercle bacilli. Bulletin of the World Health Organization. 1974;51(1):71–82. [PMC free article] [PubMed] [Google Scholar]
9.Rachow A, Ivanova O, Wallis R, Charalambous S, Jani I, Bhatt N, et al. TB sequel: incidence, pathogenesis and risk factors of long-term medical and social sequelae of pulmonary TB—a study protocol. BMC Pulm Med. 2019;19(1):4. doi: 10.1186/s12890-018-0777-3 [DOI] [PMC free article] [PubMed] [Google Scholar]
10.Stop TB. Laboratory Diagnosis of Tuberculosis by Sputum Microscopy 2013. [Available from: https://stoptb.org/wg/Gli/assets/documents/TB%20MICROSCOPY%20HANDBOOK_FINAL.pdf [Google Scholar]
11.T A. Auramine-Rhodamine Fluorochrome Staining: Principle, Procedure, Results and Limitations 2015. [Available from: http://microbeonline.com/auramine-rhodamine-fluorochrome-staining-principle-procedure-results-limitations/. [Google Scholar]
12.Division CT. Revised National TB Control Programme Training Manual for Mycobacterium tuberculosis Culture & Drug susceptibility testing 2009. [Available from: https://tbcindia.gov.in/WriteReadData/l892s/6995271860Training%20manual%20M%20tuberculosis%20C%20DST.pdf. [Google Scholar]
13.Kamerbeek J, Schouls L, Kolk A, Van Agterveld M, Van Soolingen D, Kuijper S, et al. Simultaneous detection and strain differentiation of Mycobacterium tuberculosis for diagnosis and epidemiology. Journal of clinical microbiology. 1997;35(4):907–14. doi: 10.1128/jcm.35.4.907-914.1997 [DOI] [PMC free article] [PubMed] [Google Scholar]
14.Beste DJ, Espasa M, Bonde B, Kierzek AM, Stewart GR, McFadden J. The genetic requirements for fast and slow growth in mycobacteria. PloS one. 2009;4(4):e5349. doi: 10.1371/journal.pone.0005349 [DOI] [PMC free article] [PubMed] [Google Scholar]
15.Rivas C, Coitinho C, Dafond V, Corbo M, Baldjian M. Performance of the Ogawa-Kudoh method for isolation of mycobacteria in a laboratory with large-scale workload. Revista Argentina de microbiologia. 2010;42(2):87–90. doi: 10.1590/S0325-75412010000200003 [DOI] [PubMed] [Google Scholar]
16.Jaspe RC, Rojas YM, Flores LA, Sofia Toro E, Takiff H, de Waard JH. Evaluation of the Kudoh swab method for the culturing of Mycobacterium tuberculosis in rural areas. Tropical medicine & international health: TM & IH. 2009;14(4):468–71. doi: 10.1111/j.1365-3156.2009.02236.x [DOI] [PubMed] [Google Scholar]
17.Carla M. Madeira KIA, Daisy N Sato, Celso Khosa, Nilesh Bhatt Sofia O. Viegas. Evaluation of the Ogawa-Kudoh method for tuberculosis isolation in two health units in Mozambique. African Journal of Laboratory Medicine. 2020;9(1):1–5. [DOI] [PMC free article] [PubMed] [Google Scholar]
18.Perkins MD, Roscigno G, Zumla A. Progress towards improved tuberculosis diagnostics for developing countries. The Lancet. 2006;367(9514):942–3. doi: 10.1016/S0140-6736(06)68386-4 [DOI] [PubMed] [Google Scholar]

PLoS One. doi: 10.1371/journal.pone.0300042.r001

Decision Letter 0

Padmapriya P Banada

6 Dec 2023

PONE-D-23-34900The use of Kudoh Method for culture of Mycobacterium tuberculosis and Mycobacterium africanum in The GambiaPLOS ONE

Dear Dr. Jobarteh,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by Jan 20 2024 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Padmapriya P Banada, PhD

Academic Editor

PLOS ONE

Journal requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. Note from Emily Chenette, Editor in Chief of PLOS ONE, and Iain Hrynaszkiewicz, Director of Open Research Solutions at PLOS: Did you know that depositing data in a repository is associated with up to a 25% citation advantage (https://doi.org/10.1371/journal.pone.0230416)? If you’ve not already done so, consider depositing your raw data in a repository to ensure your work is read, appreciated and cited by the largest possible audience. You’ll also earn an Accessible Data icon on your published paper if you deposit your data in any participating repository (https://plos.org/open-science/open-data/#accessible-data).

3. We suggest you thoroughly copyedit your manuscript for language usage, spelling, and grammar. If you do not know anyone who can help you do this, you may wish to consider employing a professional scientific editing service.

Whilst you may use any professional scientific editing service of your choice, PLOS has partnered with both American Journal Experts (AJE) and Editage to provide discounted services to PLOS authors. Both organizations have experience helping authors meet PLOS guidelines and can provide language editing, translation, manuscript formatting, and figure formatting to ensure your manuscript meets our submission guidelines. To take advantage of our partnership with AJE, visit the AJE website (http://learn.aje.com/plos/) for a 15% discount off AJE services. To take advantage of our partnership with Editage, visit the Editage website (www.editage.com) and enter referral code PLOSEDIT for a 15% discount off Editage services. If the PLOS editorial team finds any language issues in text that either AJE or Editage has edited, the service provider will re-edit the text for free.

Upon resubmission, please provide the following:

The name of the colleague or the details of the professional service that edited your manuscript

A copy of your manuscript showing your changes by either highlighting them or using track changes (uploaded as a *supporting information* file)

A clean copy of the edited manuscript (uploaded as the new *manuscript* file).

4. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information.

Additional Editor Comments:

Thank you for your submission. Please see the reviewers comments and concerns. Particularly, could you please highlight the differences in this study compared to your earlier published work (PMID: 28043531), since that study has not been cited in your current manuscript. Please also highlight and discuss in your discussion section how this new trial has contributed more to the field of study that your earlier study did not. Co-incidentally both studies seem to have same results. please explain.

I encourage the authors to answer the reviewers comments systematically. I hope these comments help the authors make the manuscript stronger.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Partly

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: 1. Sample size is too small (n=75), especially in case of smear negative samples (n=25). Of the 75 sputum samples collected for evaluation only 50 are smear positive and 25 are smear negative. Need to analyse a greater number of smear positive and smear negative cases.

2.Results indicate the value of the Kudoh mycobacterial method for diagnosis of pulmonary TB as a possible alternative cost-effective method for the NALC-NaOH method. However, though rapid, of the total, seven 27 (36%) samples were culture positive with both methods, fifteen 15 (20%) samples were culture positive with Kudoh method only (and negative with NALC-NaOH method) whilst twelve 12 (16%) were exclusively culture positive with NALC-NaOH method only (Table 3). So, total culture positive specimens by both methods = 35 (positive by Kudoh method) +12 (positive by NaLC-NaOH method only) =47/75. Though the Kudoh method is comparable with NaLC-NaOH method, actually combination to both these methods Kudoh + NALC-NAOH is increasing the overall culture positivity yield by 20%. Therefore, in order to detect and treat all TB cases, in resource poor settings and /or at the peripheral sites only Kudoh method can be used as a replacement to NaLC-NaOH but at sites with better resource availability Kudoh method can be used as add on method yield of TB culture positivity by nearly 20%.

3. Once AFB culture is positive, confirmation of M. tuberculosis complex using and differentiation among M. africanum colony morphology and AFB smear are not very specific and methods like spoligotyping used for species and lineage confirmation will be technically demanding and expensive to perform at resource poor settings/regions.

Reviewer #2: The authors evaluate the Kudoh method for culture of MTB and MAF in the Gambia with a focus on recovery of MAF2 isolates.

1. The conclusion that the Kudoh method can be used as an alternative to traditional LJ methods is unfounded based on the data provided in this study. While the Kudoh method showed a slightly higher culture positivity, it still missed many MTBC cultures that were (+) by LJ (and vice versa). This translates to many patients who will be culture negative if only Kudoh methods are used. While this was briefly discussed in Lines 227-232, it is suggested that the authors modify their conclusion to focus a bit more on this limitation as well as comparison to other methods of MTBC recovery and identification (Smear, MGIT, Xpert, etc).

2. To better understand the performance between the 2 methods, it is suggested to stratify the culture results based on those with either pyruvate or glycerol (either method) to see if any increase in yield for MAF (add to Table 3). Also it is suggested to break down the cultures that were positive by each method stratified by smear grade, to determine if those with lower bacillary loads had a lower positivity on one platform vs another.

3. The paper focuses on diagnostic yield of Kudoh as well as recovery of MAF. With this in mind, the Patient characteristics as well as information concerning baseline and follow-up cultures are not needed (initially discussed in Lines 164-165, yet not in the introduction). Suggest Table 1 and 2 are re-worked to include the information RE: smear grade stratification noted in Question 2 above.

4. Note the following:

Line 15: Add "S" to State

Line 27: MAF2 should be defined or spelled out (first time in paper)

Lines 87-88: Last sentence in paragraph should be reworded, or a reference provided

Line 114: The technician was blinded to what? Please clarify.

Line 151: Please re-word, summarizing the reference noted here.

Lines 180-183: Please review and move these sentences to the appropriate sections (Materials and methods, etc)

Lines 185-187: Remove numbers "27" , "15" and "12"

Line 195: "identified"

Lines 204-205: This was not an aim of the research, please revise.

Lines 220-222: This reference found 4% of all isolates exclusively detected by either method, however the results presented here show rates at 20%(Kudoh) and 16% (NALC-NaOH) which is substantially higher. Please revise/reword.

Line 271: Reword

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

**********

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

Attachment

Submitted filename: Jobarteh et al 2016.pdf

pone.0300042.s002.pdf^{(212.6KB, pdf)}

PLoS One. 2024 Mar 27;19(3):e0300042. doi: 10.1371/journal.pone.0300042.r002

Author response to Decision Letter 0

23 Jan 2024

Rebuttal Letter for reviews to manuscript “The use of Kudoh Method for culture of Mycobacterium tuberculosis and Mycobacterium africanum in The Gambia”:

Editor Comments:

Could you please highlight the differences in this study compared to your earlier published work (PMID: 28043531), since that study has not been cited in your current manuscript. Please also highlight and discuss in your discussion section how this new trial has contributed more to the field of study that your earlier study did not. Co-incidentally both studies seem to have same results. please explain.

Response: The abstract was first submitted to the Asian-African Society of Mycobacteriology (AASM) Conference and was later approved for presentation. Regretfully, unanticipated events prevented me from physically attending the conference. Unfortunately, as the abstract was accepted, it appeared in the conference proceedings which were indexed in PubMed. This was done by the organizers without my approval, and despite my absence and the fact that I never actually presented. I recognize that this may be an uncommon situation, but I would like to reassure you that the material in our submitted manuscript is unique and hasn't been released anywhere else.

Review Comments to the Author

Response: We acknowledge the small sample size as a limitation and suggest in our “Limitation” section, that prospective, large scale studies need to be conducted in the future.

Response: We agree with your suggestion, and we mentioned that besides Kudoh’s advantage for field culturing, a second advantage is to use it as add-on method. We mentioned this several times in “Discussion” and “Conclusion” of the manuscript now.

Response: It was not our intention to suggest that spoligotyping shall be done within routine diagnostic work to differentiate between MAF and MTB, for the very reasons the reviewer pointed out. We conducted spoligotyping in the present research study, to be able to differentiate between MAF and MTB, and to be able to compare the performance of the Kudoh method in between these two lineages in a MAF-endemic country. However, for clinical diagnosis and patient management in (peripheral) health centres, where Kudoh field culturing could now be beneficial, it is not needed to distinguish MTBC further between MAF and MTB with spoligotyping. We clarified this in the manuscript.

Reviewer #2: The authors evaluate the Kudoh method for culture of MTB and MAF in the Gambia with a focus on recovery of MAF2 isolates.

Response: That we suggested to replace LJ with Kudoh was a misunderstanding, and was not the intention of our statement. In order to improve our message, we modified the Discussion and Conclusion (also in line with reviewer 1’s comments) and hope that it is clearer now. The major statements that we wanted to make were (i) that Kudoh and LJ perform comparably (yet might detect different samples), (ii) that Kudoh could be a field culturing method (which cannot be done with LJ, due to all the laboratory equipment needed), and (iii) that it could be a complementary “add-on” culturing method to LJ, to increase overall culture positivity. We also expanded the discussion to mention the other TB diagnostics.

Response: The data to stratify culture results based on those with either pyruvate or glycerol is unfortunately not available. Although we grew each sample on the different slants, we only documented culture-positivity as the main read-out (without specifically documenting whether it was on pyruvate or glycerol). Stratification by smear grade was done, I hope is better now. We added this as a limitation in the respective section.

Response: The change was done.

4. Note the following:

Line 15: Add "S" to State

Response: Added S.

Line 27: MAF2 should be defined or spelled out (first time in paper)

Response: This was done.

Lines 87-88: Last sentence in paragraph should be reworded, or a reference provided

Response: The last sentence was removed.

Line 114: The technician was blinded to what? Please clarify.

Response: The technician was blinded from patients’ information like names, place of residence, etc.

Line 151: Please re-word, summarizing the reference noted here.