Diagnosis of Murine Typhus by Serology in Peninsular Malaysia: A Case Report Where Rickettsial Illnesses, Leptospirosis and Dengue Co-Circulate

Round 1

Reviewer 1 Report

The case report by Yuhana et al. describes a case of murine typhus in Malaysia diagnosed by serology.  This case report would be useful to raise awareness of murine typhus as a potential cause of febrile illness in this region.  The case section is well written, but the introduction and discussion sections need attention and polishing in regards to word usage and grammar.  Below are some suggestions for the authors’ consideration. 

 

Line 35:  suggest changing to “caused by the obligately intracellular”

 

Line 38:  It is not clear what gold standard refers to here (gold standard serologic tests or cell culture isolation).  Suggest removing “gold standard.”

 

Line 40:  Eschar is not characteristic for murine typhus.  Suggest changing to “do not elicit an eschar, which may”

 

Line 41:  suggest change to “multiorgan dysfunction”

 

Line 42:  suggest change to “death” rather than “deaths”

 

Line 55:  Suggest removing “non beta lactam” as it is not needed to qualify the tetracyclines. 

 

Line 58:  Suggest changing “unspecific” to “undifferentiated”

 

Line 59:  Suggest changing hepatitis to “elevated hepatic transaminases”

 

Line 65:  Suggest changing “non-itchy” to “non-pruritic”

 

Line 126:  Suggest changing to “inoculation of R. typhi”

 

Line 132:  Remove the word “sample” after “sera”

 

Line 133:  Suggest changing “raised” to “increase”

 

Line 145:  The phrase extremely useful is confusing here.  Although PCR amplification offers a way to determine the rickettsial species through sequencing, in the case of murine typhus, where there is no eschar to swab or biopsy, the test is not sensitive when performed on blood. 

 

Line 146 and 147:  Needs revision for word usage. 

 

Line 151:  Suggest using “severe manifestation” rather than “complication”

 

Line 152:  Suggest changing to “in a region where other rickettsial diseases…”

 

Line 156:  Last sentence of the paragraph needs to be revised for word usage and style. 

 

Line 164:  Suggest changing to “cross reacting”

 

Line 166:  Suggest removing “manage to”

 

Line 175:  Change “generic” to “undifferentiated”

 

Line 175:  Change to “signs of”

 

Line 176:  Delete the word “infection”

Author Response

Reviewer 1.

 

1. The grammatical errors highlighted  by Reviewer 1 have been amended. 

Others are:

 

Line 145: The phrase extremely useful is confusing here.  Although PCR amplification offers a way to determine the rickettsial species through sequencing, in the case of murine typhus, where there is no eschar to swab or biopsy, the test is not sensitive when performed on blood. 

 

Response: Thank you for your comments. The sentence has been changed to: "Nucleic acid detection assays targeting specific genes of R. typhiare more sensitive than serological methods for the diagnosis of acute murine typhus infection in the first 10 days of infection".

 

2. Line 146 and 147:  Needs revision for word usage.

 

Response: This section has been changed to: "Elsewhere in southeast Asia, a region where other rickettsial diseases co-circulate, a case of murine typhus co-infection with scrub typhus has been described in Laos(28). In our case, we have ruled out the scrub typhus co-infection by using the IFA targeting the specific IgM against the pooled O. tsutsugamushi Karp, Kato, and Gilliam, TA716, and other specific PCR primers against O. tsutsugamushi".

 

Reviewer 2 Report

Authors must have the article reviewed by a person who has English as first language or someone who performs review of article before submission. 

Comments for author File: Comments.docx

Author Response

Reviewer 2.

 

1. Line 39: Response: The word generichas been changed to non-specific.

 

2. Line 46: Response: This has been rephrased to the following, "Both the Indirect immunoperoxidase (IIP) and the indirect immunofluorescence assays (IFA) are considered as the gold standard serological tests and the primary difference between the two tests lies in the method of visualizing the endpoint result by using light (IIP) and fluorescence (IFA) microscopy (11, 12)."

 

3. Line 66: Response: Reviewer questioned about the rash that may have resolved after antibiotic was given. The sentence has been rephrased to be clearer as follows, "In January 2016, a 39-year-old Indian man from Teluk Intan, Perak (Northwest of Peninsular Malaysia) complained of 10 days of fever which was associated with headache, generalized myalgia and rash over the lower limbs which were non-pruritic and only noticed during the physical examinations upon admission to hospital. There was no history of antibiotic taken during the illness period prior to the hospital admission."

 

4. Line 76: Response: The reviewer raised the possibility of syphilitic rash or Steven Johnson syndrome; hence paragraph has been rephrased as follows:  "Skin examination showed erythematous macular papular rash over the lower limbs, sparing the rest of the body sites including the palms and soles. There was no evidence  of eschar. Eyes were normal with no jaundice, periorbital edema or conjunctival redness and there were no concomitant oral ulcers presence."

 

5. Line 91: Response: This has been rephrased to the following , "C-reactive protein (CRP) and procalcitonin level (PCT) were elevated at 80.23 mg/l (reference range < 5mg/l) and 0.98 ng/ml (reference range < 0.5 ng/ml), respectively."

 

6. Line 147: Response: This has been rephrased to the following, "Nucleic acid detection assays targeting specific genes of R. typhiis more sensitive for diagnosing acute murine typhus infection than by serological methods especially in the first 10 days of infection."

 

7. Line 163: Response: PCT in the murine case studies by Afzal et al. the reviewer asked about the median / mean level from that paper cited and the used pf PCT than CRP in diagnosing typhus. The author of that paper has responded to my email saying:It is not important to mention mean or median value of PCT in typhus patients, instead we want to show that PCT was elevated in most of our patients.

 

Response: line 163 rephrased to the following, "In this case, the admission PCT was performed before antibiotic treatment had  commenced and demonstrated a mildly raised level similar to previous studies (21, 30). This is the second murine typhus case that has described PCT level as one of the indicative biomarkers with the first case describing a series of murine typhus patients with PCT level of > 0.5 ng/ml upon admission however no mean or median value was provided. (21). To our knowledge, there is no information on the PCT levels during the acute and convalescence phase of this disease. We did not have a repeat PCT level following the antibiotic treatment, but a decrease in the biomarker level following a successful treatment would be reasonably expected. As PCT is a more specific biomarker for infection than CRP (the latter is considered an inflamatory marker), it should be the agent of choice in managing any infective cases (31)."

 

8. Line 168 – reviewer asks to mention about issues with cell culture and possibility of incurring extra costs

 

Response: Thank you very much for your suggestion. We have added a section as follows:

"…hence a clear grading approach in diagnosis of rickettsial infections using combinations of culture, antigen – or DNA detection coupled with dynamic serology have proven to be useful (29). However, the three-pronged approach is not feasible in most clinical settings as in vitro rickettsial isolation requires laborious cell culture technique and potentially require BSL-3 laboratory containment (based on the risk-assessment) that may lead to highdiagnostic costs."

Reviewer 3 Report

1. Please read through the manuscript and make necessary spelling and grammar corrections. For example, line 46.

2. The IFA titer is presented as > 3,200 and > 12,800 for acute and convalescent samples, respectively. While the titer is probably high enough but this presentation may mislead to a conclusion that the end titers of acute and convalescent samples were not determined (e,g, acute sample could have end titer of 6,400 but the covalescent is only 12,800) and may not really fulfill the 4-fold titer increase criterion. The authors may consider to report the end titer (if it was performed) to eliminate the potential confusion.

3. It may be beneficial the authors emphasized the importance of proper treatment in this particular case report. While ceftriaxone was prescribed to treat leptospirosis as it was the suspected disease, in terms of patient management, it may be better to recommend doxycycline as the first treatment option (as it is appropriate) to in areas where these leptospirosis, scrub typhus and murine typhus are known co-circulating.

4. What is the reason for overlapping treatment of both doxycycline and ceftriaxone?

Author Response

Reviewer 3.

1. Line 109 àThe IFA titer is presented as > 3,200 and > 12,800 for acute and convalescent samples, respectively. While the titer is probably high enough but this presentation may mislead to a conclusion that the end titers of acute and convalescent samples were not determined (e,g, acute sample could have end titer of 6,400 but the covalescent is only 12,800) and may not really fulfill the 4-fold titer increase criterion. The authors may consider to report the end titer (if it was performed) to eliminate the potential confusion.

 

Response: The sentence has been rephrased to the following, "The admission serum showed a Murine typhus IgM result of 1:3200 in the IFA which later increased to 1:12800 in the convalescent sera which was collected five days later (Figure 1)".

 

2. It may be beneficial the authors emphasized the importance of proper treatment in this particular case report. While ceftriaxone was prescribed to treat leptospirosis as it was the suspected disease, in terms of patient management, it may be better to recommend doxycycline as the first treatment option (as it is appropriate) to in areas where these leptospirosis, scrub typhus and murine typhus are known co-circulating.

 

Response: Hence in line 167 has been changes to the following, "As doxycycline has been shown to be an effective antibiotic for both mild leptospirosis and rickettsial infections such as scrub typhus and murine typhus, we suggest that in an area where the zoonotic infections co-circulate, doxycycline should be considered as the first line antibiotic agent in patients with mild infection (15, 32)." 

 

4. What is the reason for overlapping treatment of both doxycycline and ceftriaxone?

 

Response: In this case there was no overlapping of treatment with the two antibiotics. 

Hence in line 103 has been changed to the following, "The intravenous ceftriaxone was then stopped and oral doxycycline 200mg was then promptly administered and maintained on 100mg twice a day for 7 days (Figure 1)."

 

Reviewer 4 Report

Thank you for the opportunity to review this significant piece of work.

The authors tried to diagnose the case as murine typhus according to the serological result because the PCR assays were negative.

 

Major comment 1.

 

Why did not the authors use only IgM against R. typhi?

As the authors mentioned, there is serological cross-reactivity between spotted fever groups and murine typhus. Although they obtained the high-titer IgM, the current case is hard to diagnose as murine typhus because of the cross-reactivity to the spotted fever group. Since the authors have a paired sample, I recommend testing IgG against R. typhi and other spotted fever groups pathogen, in addition to IgM.

 

Major comments 2.

 

I disagree that rule-out of scrub typhus infection because of only relies on the result of IgM IFA. In the endemic area, the possibility of “re-infection” of scrub typhus should be considered. IgM and PCR may have lower sensitivity in the re-infection case and the serological reactivity has quite different from primary infection. And the authors should be including IgG result to insist on rule out of scrub typhus infection.

 

 

Minor comments:

 

Minor comment 1.

Line 36: Add a period after ”Xenopsylla cheopis”

 

Minor comment 2.

Figure 1: Why the body temperature was different between figure 1(37.5 degrees on Day 10) and “Line 73” (oral temperature of 39 degrees)?

 

Author Response

Reviewer 4.

Major comment 1.

Why did not the authors use only IgM against R. typhi? As the authors mentioned, there is serological cross-reactivity between spotted fever groups and murine typhus. Although they obtained the high-titer IgM, the current case is hard to diagnose as murine typhus because of the cross-reactivity to the spotted fever group. Since the authors have a paired sample, I recommend testing IgG against R. typhi and other spotted fever groups pathogen, in addition to IgM.

 

Response: Amended as suggested with additional testing. We have tested the samples forspotted fever and scrub typhus IgM and IgG and R typhi IgG and have included the resukts in the manuscript.The passage now reads as follows,  "The admission serum showed a Murine typhus IgM result of 1:3200 and IgG 1:800 in the IFA which later increased to IgM 1:12800 and IgG 1:800 in the convalescent sera which was collected five days later (Figure 1). Comparatively, IFAs for scrub typhus IgM and IgG were <1:100 for both samples and spotted fever group rickettsia IFA gave IgM result of 1:100 and IgG <1:100 and IgM 1:800 and IgG <1:100 in the convalescent sample."

 

Major comments 2.

I disagree that rule-out of scrub typhus infection because of only relies on the result of IgM IFA. In the endemic area, the possibility of “re-infection” of scrub typhus should be considered. IgM and PCR may have lower sensitivity in the re-infection case and the serological reactivity has quite different from primary infection. And the authors should be including IgG result to insist on rule out of scrub typhus infection

 

Response: Amended. Please  see the additional serology results above which demonstrates no evidence of STG infection. 

 

Line 152 has been made more subtle  and reads as follows, "Elsewhere in south East Asia, a region where other rickettsial diseases co-circulate, a case of murine typhus co-infection with scrub typhus has been described in Laos (28). In our case, we have ruled out the scrub typhus co-infection by using the IFA targeting the specific IgM against the pooled O. tsutsugamushi Karp, Kato, and Gilliam, TA716, and other specific PCR primers against O. tsutsugamushi." 

 

Also Line 181, "Other major limitation in this reporting is that we could not rule out the possibility of secondary scrub typhus infection due to lack of IgG assay for scrub typhus in our diagnostic panel. This is important in term of diagnosis especially in area where scrub typhus is considered to be endemic, but the treatment outcome may not be much affected."

 

 

 

 

 

 

 

 

Round 2

Reviewer 1 Report

Line 40:  Need to revise "Do not an eschar."  The sentence is unclear as written.  

149 - 150:  Neither PCR nor serology is sensitive early in illness.  I suggest revising or not including this idea here.  

Author Response

Line 40:  Need to revise "Do not an eschar."  The sentence is unclear as written.

 

Changed to

Bites from the rat fleas do not elicit an eschar, unlike in scrub typhus or spotted fever rickettsial typhus which may assist in diagnosis.

 

149 - 150:  Neither PCR nor serology is sensitive early in illness.  I suggest revising or not including this idea here.  

 

The sentence has been removed

Reviewer 2 Report

Authors must have the article reviewed by a person who is proficient in English grammar. Also, they should carefully consider to add the grammar and punctuation changes suggested during the first review and consider to add the second review suggestions. The main point of the article is to alert the clinicians in Malaysia Peninsula that murine typhus must be in the differential diagnosis of febrile illness and they can make the diagnosis by serology, PCR and/or culture. It should be a short case.

Comments for author File: Comments.docx

Author Response

Line 163: 

PCT in the murine case studies by Afzal et al. the reviewer asked about the median / mean level from that paper cited and the used pf PCT than CRP in diagnosing typhus. The author of that paper has responded to my email saying: It is not important to mention mean or median value of PCT in typhus patients, instead we want to show that PCT was elevated in most of our patients. It is not expected PCT to be elevated in cases on infection in general (not only murine typhus)? I do not think there is any study looking the mean or median of PCT for murine typhus because it is not specific for murine typhus and it will be elevated as it is for any other infection and is not more sensitive or specific for murine typhus, The advantage of PCT on murine typhus is to tell that patient has a bacterial infection instead a viral infection, nothing else.

 

The sentence has been changed to

This case had demonstrated a raised in PCT level similar to previous study by Afzal et al., which showed raised PCT level of > 0.5 ng/ml in his cohort. As PCT is a more specific biomarker for infection than CRP (the latter is considered an inflammatory marker), it should be the agent of choice in managing infective cases including as a guide to treatment response.

Reviewer 4 Report

The manuscript has made considerable changes and addressed the queries well.

I have some comments for the manuscript which has changed.

 

Major comments.

 

1.       Is that true that the authors stated as “IFAs for scrub typhus IgM and IgG were <1:100 for both samples” in line 111-112? On the other hand, the authors stated as “we could not rule out the possibility of secondary scrub typhus infection due to lack of IgG assay for scrub typhus our diagnostic panel” in line 181-182. Which description is correct? Did the authors use IgG? It seems to be inconsistent.

 

2.       The authors need to check if the references match the sentence.

 

Line 40: Ref 3. reported about the diagnosis for scrub typhus which is mite-borne infection, not about rat flea-borne infection. According to Ref No. 4, eschar was rarely found in both patients with scrub typhus and murine typhus. Please check the reference again and address clearly.

Also,  What did “do not an eschar” mean? 

 

Line 146-147: “IgM against R. typhi is invariably absent in the first week of illness, thus a paired sera sample is required to demonstrate a four-fold raised in the IgM to demonstrate a robust evident of acute infection (18, 26).”

 

   -> Ref No. 18 stated only about the diagnosis of scrub typhus, not about murine typhus.

 

Line 149-151: “Nucleic acid detection assays targeting specific genes of R. typhi are more sensitive than serological methods for the diagnosis of acute murine typhus infection in the first 10 days of infection (18, 27).”

 

-> Ref No. 18 stated only about the diagnosis of scrub typhus, not about murine typhus.

 

Minor comments.

 

Line 46: “Indirect immunoperoxidase” should be changed as “indirect immunoperoxidase.”

   

Line 109: “Murine typhus” should be changed as “murine typhus”.?

 

Line 112: “spotted fever group rickettsia IFA”

->  Please address which rickettsia was used, specifically.

 

Line 147: “a robust evident” should be changed as “a robust evidence.”

 

 

 

Author Response

Is that true that the authors stated as “IFAs for scrub typhus IgM and IgG were <1:100 for both samples” in line 111-112? On the other hand, the authors stated as “we could not rule out the possibility of secondary scrub typhus infection due to lack of IgG assay for scrub typhus our diagnostic panel” in line 181-182. Which description is correct? Did the authors use IgG? It seems to be inconsistent.

 

The sentence “Other major limitation in this reporting is that we could not rule out the possibility of secondary scrub typhus infection due to lack of IgG assay for scrub typhus in our diagnostic panel. This is important especially in area where scrub typhus is considered to be endemic” has now been removed as it was overlooked in the previous correction.

Line 146-147: “IgM against R. typhi is invariably absent in the first week of illness, thus a paired sera sample is required to demonstrate a four-fold raised in the IgM to demonstrate a robust evident of acute infection (18, 26).”

 Ref No. 18 stated only about the diagnosis of scrub typhus, not about murine typhus.

The reference pertaining to the scrub typhus has been removed.

Line 149-151: “Nucleic acid detection assays targeting specific genes of R. typhi are more sensitive than serological methods for the diagnosis of acute murine typhus infection in the first 10 days of infection (18, 27).”

Ref No. 18 stated only about the diagnosis of scrub typhus, not about murine typhus.

The reference pertaining to the scrub typhus has been removed.

Line 46: “Indirect immunoperoxidase” should be changed as “indirect immunoperoxidase.”

Changed

Line 109: “Murine typhus” should be changed as “murine typhus”.?

Changed

Line 112: “spotted fever group rickettsia IFA”

R. felis IFA has been added in the sentence.

Line 147: “a robust evident” should be changed as “a robust evidence.”

Changed

 

Round 3

Reviewer 2 Report

Poor English grammar in terms of concordance. Multiple sentences need to be re-phrased to make sense. No value of PCT for diagnosis of murine typhus. 

Comments for author File: Comments.pdf

Author Response

please check the attachment.

Author Response File: Author Response.pdf

Reviewer 4 Report

The manuscript has improved.

Author Response

thanks