Discussion
This analysis indicates that microscopically-determined microfilaraemia is higher in CAP blood samples than in VEN blood samples for L. loa infection and M. perstans infection. Previous studies show concordant evidence in favour of higher L. loa and M. perstans microfilaraemia in CAP than in VEN blood samples when equal blood volumes were evaluated. In 1950, Kershaw assessed CAP and VEN blood samples of each 50 microliters blood taken from two participants and demonstrated a higher microfilarial blood density in CAP blood than in VEN blood [17]. Later, in 1990 Noireau and Apembet assessed the diagnostic sensitivity of 40 microliters of CAP blood samples in 39 and 97 individuals infected with L. loa and M. perstans, respectively whose infection status was ascertained by light microscopical assessment of 5ml VEN blood lysed with 2% saponin [18]. They indicated that 40 microliters of CAP blood had a sensitivity of 67% (26/39) and 62% (60/97) to detect L. loa and M. perstans, respectively. However, a direct comparison of diagnostic properties of CAP and VEN blood samples was not possible, as preparation methods and blood quantities were different prior to diagnostic assessment. Applying the exact same diagnostic methods and investigating equal volumes of blood we noted that L. loa microfilaraemia was about 33% higher in CAP blood than in VEN blood (p = 0.0027) and about 25% higher for M. perstans infection (p = 0.08). Interestingly, while the best model estimate indicates increased odds for L. loa microfilariae detection in CAP blood (OR: 1.24) compared with VEN blood, formal hypothesis testing did not reveal any evidence for such an effect in the target population (p = 0.52). Yet, as the applied parameters in our sample size calculation might have been overestimated (i.e. estimated OR: 2.5) there is a possibility that the study was underpowered to detect a true effect. However, in microscopy the probability of detection of blood parasite increases, as the parasite quantity increases and the limit of detection in this study was 100 microfilariae per one milliliter blood. That being said, the median L. loa microfilaraemia in our study sample was high both in CAP (3,650 [IQR: 275–11,100]) and in VEN blood (2,775 [IQR: 200–8,875]). Therefore, if the true population medians of CAP and VEN microfilaraemia are truly above the limit of detection in microscopy, then absolute differences in CAP-VEN microfilaraemia might not contribute to a differential odds of microfilaria detection. On the other hand, there was an increased odds for M. perstans microfilariae detection in CAP blood compared with that in VEN blood (OR: 1.65; p = 0.041). Such increased odds of microfilaria detection in CAP blood seem plausible as median CAP and VEN M. perstans microfilaraemia was exactly at the microscopic limit of detection and therefore even slightly lower levels microfilaraemia in VEN blood can explain the increased odds for detection of microfilariae in CAP blood (i.e. +25% higher microfilaraemia in CAP blood; p = 0.08). This hypothesis is supported by the increasing CAP-VEN sensitivity difference in a sub-population of participants with a low M. perstans microfilaraemia. Additionally, the study might be underpowered to detect an absolute difference in CAP-VEN M. perstans microfilaraemia, as average values in our study sample were much lower than estimated in sample size calculations (50% estimated versus 25% observed). Furthermore, although this cannot be supported by data of our study it may also be possible that in a larger sample of participants L. loa microfilaraemia might be more often detected in CAP than in VEN blood, given the relatively higher values for microfilaraemia in CAP blood relative to those in VEN blood. However, it is of mention that to the best of our knowledge this is the largest study to date on this topic.
Traditionally, infection with L. loa received mainly attention in areas of onchocerciasis co-endemicity, as high L. loa microfilaraemia can lead to potentially lethal encephalopathy during administration of ivermectin (i.e. the treatment used in mass drug administration campaigns against onchocerciasis). Consequently, it is recommended that L. loa microfilaraemia needs to be below a certain threshold before treatment with ivermectin and other strong microfilaricidal agents can be safely administered. Therefore, as for other tropical pathogens the quantification of L. loa microfilaraemia and knowledge of pre-analytical influences such as those by CAP and VEN blood sampling are important [25,26]. For patient safety, it might therefore be beneficial if microfilaraemia levels are below the given target threshold in CAP blood, based on findings of this study, that CAP microfilaraemia levels are on average about 33% higher than those in VEN blood. In the past, the implementation of ivermectin-based mass drug administration for the control of onchocerciasis or lymphatic filariasis has been impaired because of afore-mentioned serious adverse events in persons with a high L. loa microfilaraemia (i.e. > 20,000 microfilariae/milliliter blood) [27]. To overcome this treatment gap, a so called ‘Test and not treat’ strategy was developed which is based on withholding mass drug administration from those people with high levels of L. loa microfilariae [28,29]. Such highly microfilaraemic persons are identified by means of rapid testing with a diagnostic tool named LoaScope, which analyses samples of peripheral blood [28,30]. Therefore, in order to ensure safety of ivermectin mass drug administration CAP blood should be used for rapid quantification of L. loa. microfilariae with LoaScope. It is likely that CAP blood sampling may already constitute the main blood sampling method for rapid assessments, as it is an easy procedure even under field conditions. However, besides this feasibility aspect this current study adds an additional safety argument in favour of CAP blood sampling.
Generally, studies on antimicrobial chemotherapy often rely on the exact quantification of microbial blood density in study participants. Therefore, findings of this present study should be considered for protocols of studies on the therapy of loiasis and mansonellosis. Additionally, in spite of evidence for a higher average microfilaraemia in CAP blood versus VEN blood regarding infections with L. loa and M. perstans, highly favourable intraclass correlation coefficients demonstrate that a CAP microfilaraemia is well reproducible in VEN blood and vice versa.
In 1917, Yorke and Blacklock discussed that microfilariae of Wuchereria bancrofti are more abundant in CAP than in VEN blood irrespective of varying microfilarial peak densities during a 24-hour cycle. This phenomenon was explained by mechanical obstruction of microfilariae during passage through the small cutaneous capillary blood vessels aiding in the piling up of larvae in the cutaneous vessels [9]. It is likely that this explanation also applies to microfilariae of L. loa and M. perstans which are similar in size (about 275x7μm and 210x4μm, respectively) [1,2]. Furthermore, from an evolutionary-biological perspective a high quantity of microfilariae in CAP blood seems plausible, as CAP blood is taken up during a blood meal by the insect vector [31]. Thereby, a high CAP microfilaraemia facilitates favourable chances for transmission of the infection to the insect-vector and might consequently contribute to the survival of the respective parasite species.
It needs to be mentioned, that the probability of microfilaraemia detection by light microscopy does not only potentially depend on the type of blood sample (i.e. CAP or VEN) used for diagnostic purposes, but also on the quantity of the investigated sample volume. This study only investigated 10 microliters of blood, a blood quantity which is common particularly for diagnosis of malaria in clinical and research settings. This constitutes a limitation, as it is possible to extract higher quantities of CAP blood during a given CAP blood sampling session (e.g. 50 microliters). Furthermore, in individual case management when infection by a filarial blood parasite is suspected it is recommended to withdraw venous blood of larger quantities (i.e. several milliliters) for microfilariae detection at different days [7,18]. Yet, it was the objective of this study to investigate CAP-VEN differences in microfilaraemia and a potentially resulting differential odds for microfilaraemia-detection. For that it was important to investigate equal sample volumes of CAP and VEN blood in order to enable a fair comparison. A quantity of 10 microliters was considered as feasible to be assessed in sufficient detail by several microscopists. The applied two-to-three microscopist approach is a methodological strength and ensures exact quantification of microfilaraemia so that measurement errors are considered minimal. Furthermore, it is a strength that all microscopists were blinded to each other’s results and received training on the diagnostic features of the two target microfilariae of L. loa and M. perstans.
10.1371/journal.pntd.0009623.r001
Decision Letter 0
Henao-Martínez
Andrés F.
Deputy Editor
2021
Andrés F. Henao-Martínez
This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Submission Version
0
5 May 2021
Dear MD Mischlinger,
Thank you very much for submitting your manuscript "Diagnostic performance of capillary and venous blood samples in the detection of Loa loa and Mansonella perstans microfilaraemia using light microscopy" for consideration at PLOS Neglected Tropical Diseases. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. In light of the reviews (below this email), we would like to invite the resubmission of a revised version that takes into account the reviewers' comments.
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Sincerely,
Andrés F. Henao-Martínez, M.D.
Deputy Editor
PLOS Neglected Tropical Diseases
***********************
Reviewer's Responses to Questions
Key Review Criteria Required for Acceptance?
As you describe the new analyses required for acceptance, please consider the following:
Methods
-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?
-Is the study design appropriate to address the stated objectives?
-Is the population clearly described and appropriate for the hypothesis being tested?
-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?
-Were correct statistical analysis used to support conclusions?
-Are there concerns about ethical or regulatory requirements being met?
Reviewer #1: The objective of the study conducted by Mischlinger et al. was to compare the number of Loa loa and Mansonella perstans microfilariae observed in 10 µL of venous blood and 10 µL of capillary blood. The authors found that the microfilarial densities (MfD) were significantly higher in capillary blood and state that this finding is important because it “might have implications for treatment algorithms of onchocerciasis and loiasis, in which exact quantification of L. loa microfilaraemia is of importance”.
The authors say their study is the first to be conducted to compare Loa and M. perstans MfDs in venous and capillary blood. This is not correct. Kershaw (Ann Trop Med Parasitol 1950) studied this on two subjects (and found that the L. loa and M. perstans MfDs are higher in capillary blood). Similarly, Noireau et al. (J Trop Med Parasitol 1990) compared L. loa and M. perstans MfDs in venous and capillary blood collected from 201 subjects. In addition, the fact that the MfD is higher in capillary blood than in venous blood is also very well known for Wuchereria bancrofti, the cause of lymphatic filariasis, whose microfilariae are similar in size to those of L. loa. It is very strange that the authors did not review the studies comparing different methods used to quantify W. bancrofti MfD from venous and capillary blood. One of the first articles is that of Yorke & Blacklock, Ann Trop Med Parasitol 1917. Over 15 other articles present data on this subject. This review should have been included in the introduction or in the discussion section of the paper.
The authors collected 10 µL of capillary blood and 10 µL of venous blood (without fully explaining how they proceeded for the venous blood). As they write themselves, this quantity is very low but they explain that it "was restricted by the small extractable quantities of CAP blood". This argument does not hold since the volume of blood routinely (and easily) taken by finger prick during filariasis surveys is 50 µL. It would have been more interesting to compare the MfDs in two 50 µL samples, which would have detected more subjects with low microfilaremia.
The statistical methods used to analyse the data are relatively complex. The authors write rightly that the logarithm of 0 does not exist but it is common in the field of filariasis to add 1 to the MfD to be able to calculate geometric means taking into account subjects with negative results (Williams’ mean).
Reviewer #2: The objectives were clearly stated and the study design was appropriate, the study population was also clearly described and appropriate though the adequate sample size was not achieved to ensure adequate power (acknowledged by the researchers)
Ethical statement, should state the ethics committee approval number.
There are problems with clarity especially in the methods and results section for non-specialists. The authors need an English specialist to review that section and correct some grammatical presentations.
Reviewer #3: (No Response)
--------------------
Results
-Does the analysis presented match the analysis plan?
-Are the results clearly and completely presented?
-Are the figures (Tables, Images) of sufficient quality for clarity?
Reviewer #1: The analysis presented match the analysis plan and the results are clearly and completely presented.
Reviewer #2: Yes the analysis presented match the analysis plan but the prose of the results needs to be made clearer. On the other hand, the tables are clearly presented.
Reviewer #3: (No Response)
--------------------
Conclusions
-Are the conclusions supported by the data presented?
-Are the limitations of analysis clearly described?
-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?
-Is public health relevance addressed?
Reviewer #1: By reading the literature on W. bancrofti mentioned above, the authors could have discussed the hypotheses proposed by the various authors who have worked on the subject to explain the phenomenon of higher MfD in capillary blood. In the manuscript, they just say that in malaria the differences “can be explained by host-parasite receptor interactions”. However, it is clear that the microfilariae are large organisms (unlike Plasmodium, to which the authors refer several times) and that the observed differences are probably at least partly due to the ratios between the size of the parasites and the diameter of blood vessels.
When the authors say that their findings may have implications for the treatment of onchocerciasis and loiasis because it is necessary to know the exact Loa MfD before giving treatment, they overestimate somewhat the interest of their results. Actually, the decisions are made according to the MfD measured by the reference method which is the quantification in capillary blood (which enables to detect at-risk subjects with high MfD). The fact that the density is lower in the venous blood does not really matter.
Reviewer #2: The conclusions are supported by the presented results, and the limitations of analysis clearly described. The authors have discussed adequately the implications of the data presented and its relevnce to public health.
Reviewer #3: (No Response)
--------------------
Editorial and Data Presentation Modifications?
Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.
Reviewer #1: Line 83, delete “(L.)” and “(M.)”
Line 85, delete both “spp.”
Line 97: cite Asio et al., Parasitology Research 2009
Line 103: in the list of references, reference 8 is incomplete
Line 140: check reference number
Reviewer #2: The sample size determination should come before materials and parasitological analysis, not after statistical considerations.
Reviewer #3: (No Response)
--------------------
Summary and General Comments
Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.
Reviewer #1: This paper is of limited interest. The results confirm those presented in other papers on Loa loa, M. perstans, and W. bancrofti. They are not very original and will not have any implication on the strategies to combat onchocerciasis in loiasis-coendemic areas.
Reviewer #2: The study adds some value to the body of knowledge already known in this area. Identifying that capillary blood samples yield higher levels of parasitaemia has been known for malaria, but not for microfilaria. That is what this study fills in.
The details of the methodology are sufficient to reproduce the study
There are problems with clarity especially in the methods and results section for non-specialists. The authors need an English specialist to review that section and correct some grammatical presentations. Major ones have been highlighted here. The sample size determination should come before materials and parasitological analysis, not after statistical considerations.
• The academic achievements should be removed from the authors names
• Line 117, ‘two thick smears of blood, 10 microliters each were prepared per participants’
• Line 118, ‘one from capillary and one from venous blood at the same time’
• Line 162, ‘120 participants each with L. loa infection and 120 with M. perstans infection are required’
• Ethical statement, should state the ethics committee approval number
• Line 170, participants were
• Line 172, subsequent 10-year age group
• Line 182 remove revealed
• Line 282 largest study to date
• Information in line 303, is better put in the methods section
• Lines 327, 338, the references are incomplete
Reviewer #3: The paper by Mischlinger et al. presents the results of comparing measurements of microfilariae of Loa loa and Mansonella perstans between venous and capillary blood collected from 201 patients.
The paper presents an incomplete bibliography as there are two papers that have already performed this experiment; and a large number of papers on Wuchereria bancrofti. The authors state that this is the first experiment, which is not the case. The authors should complete the bibliography accordingly. Instead, the comparison with malaria is throughout the paper, which is less coherent.
The authors should clarify in which framework this study was done. Indeed, it is obvious that this is an ancillary study and that it was not prepared for this purpose. In particular, this would explain why only 10 μL were collected from both venous and capillaries. It would be good to clarify this, and to put less emphasis on possible technical difficulties, as otherwise readers not used to this disease might think that it is difficult to perform capillary or venous sampling, which is not the case.
Last, it seems that the authors should more discuss about the raison about the observed difference.
Finally, the authors indicate that these results could have implications for control programmes, but this is difficult to relate. The samples used are always capillary, and the thresholds have been defined for this type of sampling, so, in my opinion, these results have no obvious implications.
In the end, although the analyses are well done, the paper may not be of sufficient interest for publication in Plos NTD.
--------------------
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Reviewer #2: No
Reviewer #3: No
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10.1371/journal.pntd.0009623.r003
Decision Letter 1
Henao-Martínez
Andrés F.
Deputy Editor
2021
Andrés F. Henao-Martínez
This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Submission Version
1
3 Jul 2021
Dear MD Mischlinger,
We are pleased to inform you that your manuscript 'Diagnostic performance of capillary and venous blood samples in the detection of Loa loa and Mansonella perstans microfilaraemia using light microscopy' has been provisionally accepted for publication in PLOS Neglected Tropical Diseases.
Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.
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IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. Requests for major changes, or any which affect the scientific understanding of your work, will cause delays to the publication date of your manuscript.
Should you, your institution's press office or the journal office choose to press release your paper, you will automatically be opted out of early publication. We ask that you notify us now if you or your institution is planning to press release the article. All press must be co-ordinated with PLOS.
Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases.
Best regards,
Andrés F. Henao-Martínez, M.D.
Deputy Editor
PLOS Neglected Tropical Diseases
Andrés Henao-Martínez
Deputy Editor
PLOS Neglected Tropical Diseases
***********************************************************
Reviewer's Responses to Questions
Key Review Criteria Required for Acceptance?
As you describe the new analyses required for acceptance, please consider the following:
Methods
-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?
-Is the study design appropriate to address the stated objectives?
-Is the population clearly described and appropriate for the hypothesis being tested?
-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?
-Were correct statistical analysis used to support conclusions?
-Are there concerns about ethical or regulatory requirements being met?
Reviewer #1: Yes for all questions except the last
No ethical concern
Reviewer #2: (No Response)
**********
Results
-Does the analysis presented match the analysis plan?
-Are the results clearly and completely presented?
-Are the figures (Tables, Images) of sufficient quality for clarity?
Reviewer #1: Yes
Reviewer #2: (No Response)
**********
Conclusions
-Are the conclusions supported by the data presented?
-Are the limitations of analysis clearly described?
-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?
-Is public health relevance addressed?
Reviewer #1: Yes
Reviewer #2: (No Response)
**********
Editorial and Data Presentation Modifications?
Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.
Reviewer #1: No
Reviewer #2: Accept
**********
Summary and General Comments
Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.
Reviewer #1: No comments
Reviewer #2: Suggested revisions have implemented, manuscript can be accepted
**********
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Reviewer #1: No
Reviewer #2: No