Pan-Echinocandin-Resistant Candida glabrata Bloodstream Infection Complicating COVID-19: A Fatal Case Report

Round 1

Reviewer 1 Report

The authors describe a case of COVID-19 pneumonia in a 79 year old patient who developed a series of nosocomial infections during a protracted hospital treatment course with a fatal outcome. The patient had risk factors for more severe COVID-19 infection including types 2 diabetes mellitus. After two successfully treated episodes of bacterial sepsis, he developed candidemia on Day 35 due to Candida glabrata which was treated with caspofungin but candidemia recurred while he was still on caspofungin. In vitro susceptibility testing revealed that the parental C. glabrata isolate was susceptible to echinocandins whereas  one of two morphotypes from the recurrent isolate   culture was resistant and further analysis revealed mutations in FKS gene hot spots 1 and 2 linked to echinocandidn resistance. Typing analysis using Maldi-TOF MS suggested these two isolates were closely related and may be the same strain. Therefore the presumption is that resistance emerged in vivo following exposure of the C. glabrata to caspofungin.

Overall, the case is well presented and the table and 2 figures are appropriate giving further detail on the results.

The authors  are hesitant to suggest that the candidemia was the immediate cause of death as this was a complex case and his death was immediately preceded by a surgical intervention which may also be relevant to the outcome

It would be helpful to know the actual date of admission to hospital which would put the patient's initial management into context with regard to his COVID19 treatment.

Page 1:  line 39 insert "in" after contributing to further increase....

Page 2: line 53 please clarify what "stadium IV" means.

same page line 63 tense changes. I suggest keep all discussion to past tense. So on line 65 change sentence to (which was continued for the next five days).

line 75 " He apparently improved is a bit vague, either he improved or he didn't.

line 90 replace "unvaried" to unchanged

Page 3: line 123 remove "the" to now read to amphotericin B

line 128 Please explain the Maldi TOF a bit better. which system was used, i.e. in house or commercial. What was the origin of the additional 17 C glabrata isolates analysed? Were some, as appears to be the case in Figure 1 also paired isolates?

Can the authors add that next generation sequencing, which is nowadays feasible, could have provided greater discrimination/evidence of similarity.

What if any, was the relationship of the candidemia to the presence of indwelling vascular catheters? did the patient have vascular line in at the time of the first episode of candidemia, and was it removed promptly after the diagnosis of candidemia was known?

Page 6: line 36 change sentence to "....in our case as in  other cases.

line 39 Sentence starting "However, diabetes..." is a bit unclear as to its meaning. Rather than saying "are in an indiscernible balance" perhaps change to "commonly co-exist during patients' ICU stay"

Page 7 line line 55 sentence starting " In two reports..." is not clear, please review.

line 67 insert "were" after of which 6...

line 86 insert "by" after resistance

Lines 92/93 transpose " critical patient's condition" to patient's critical condition.

Line 97 change "being" to when

Author Response

The authors describe a case of COVID-19 pneumonia in a 79-year-old patient who developed a series of nosocomial infections during a protracted hospital treatment course with a fatal outcome. The patient had risk factors for more severe COVID-19 infection including types 2 diabetes mellitus. After two successfully treated episodes of bacterial sepsis, he developed candidemia on Day 35 due to Candida glabrata, which was treated with caspofungin, but candidemia recurred while he was still on caspofungin. In vitro susceptibility testing revealed that the parental C. glabrata isolate was susceptible to echinocandins whereas one of two morphotypes from the recurrent isolate culture was resistant and further analysis revealed mutations in FKS gene hot spots 1 and 2 linked to echinocandin resistance. Typing analysis using Maldi-TOF MS suggested these two isolates were closely related and may be the same strain. Therefore, the presumption is that resistance emerged in vivo following exposure of the C. glabrata to caspofungin.

Overall, the case is well presented and the table and 2 figures are appropriate giving further detail on the results.

The authors are hesitant to suggest that the candidemia was the immediate cause of death as this was a complex case and his death was immediately preceded by a surgical intervention, which may also be relevant to the outcome.

Answer: To this regard, we added a sentence to underline that candidemia as an immediate cause of death in our patient remains a matter of debate. See line 7, lines 35 to 37 of the revised manuscript.

It would be helpful to know the actual date of admission to hospital, which would put the patient's initial management into context with regard to his COVID19 treatment.

Answer: As suggested, we added the date of admission to hospital. See page 2, line 64 of the revised manuscript.

Page 1: line 39 insert "in" after contributing to further increase....

Answer: As suggested, we added “in” after “contributing to further increase...”. See page 1, line 40 of the revised manuscript.

Page 2: line 53 please clarify what "stadium IV" means.

Answer: As required, we added a specification about the “stadium IV” stage of PAD disease. See page 2, lines 68 to 69 of the revised manuscript.

Same page line 63 tense changes. I suggest keep all discussion to past tense. So on line 65 change sentence to (which was continued for the next five days).

Answer: As suggested, we changed the verb tense on line 63 and, consistently, the sentence on line 65. See page 2, lines 79 and 81 of the revised manuscript.

Line 75 "He apparently improved is a bit vague, either he improved or he didn't.

Answer: We changed the sentence to “He improved”. See page 2, line 91 of the revised manuscript.

Line 90 replace "unvaried" to unchanged

Answer: We replaced “unvaried” to “unchanged”. See page 3, line 106 of the revised manuscript.

Page 3: line 123 remove "the" to now read to amphotericin B

Answer: As required, we removed “the” before “amphotericin B”. See page 3, line 139 of the revised manuscript.

Line 128 Please explain the Maldi TOF a bit better. Which system was used, i.e. in house or commercial? What was the origin of the additional 17 C. glabrata isolates analysed? Were some, as appears to be the case in Figure 1 also paired isolates? Can the authors add that next generation sequencing, which is nowadays feasible, could have provided greater discrimination/evidence of similarity?

Answer: As required, we added details about the MALDI-TOF MS based analysis as well as the origin of additional isolates included in the analysis. Additionally, we commented on that next-generation sequencing analysis could have provided greater discrimination/evidence of similarity among the isolates studied. See pages 3 and 4, lines 145 to 154 of the revised manuscript.

What if any, was the relationship of the candidemia to the presence of indwelling vascular catheters? Did the patient have vascular line in at the time of the first episode of candidemia, and was it removed promptly after the diagnosis of candidemia was known?

Answer: The patient did not have a vascular line at the time of the first episode of candidemia. Consequently, this detail was omitted in the manuscript.

Page 6: line 36 change sentence to "...in our case as in other cases.

Answer: As suggested, we changed to “in our case as in other cases”. See page 7, line 29 of the revised manuscript.

Line 39 Sentence starting "However, diabetes..." is a bit unclear as to its meaning. Rather than saying, "are in an indiscernible balance" perhaps change to "commonly co-exist during patients' ICU stay".

Answer: As suggested, we changed to “commonly coexist during patients’ hospital stay”. See page 7, line 33 of the revised manuscript.

Page 7 line 55 sentence starting “In two reports..." is not clear, please review.

Answer: As required, we modified the sentence for clarity. See page 8, lines 51 to 54 of the revised manuscript.

Line 67 insert "were" after “of which 6...”

Answer: As required, we added the term “were” after “of which 6”. See page 8, line 64 of the revised manuscript.

Line 86 insert "by" after resistance.

Answer: As required, we added the term “by” after “resistance”. See page 8, line 85 of the revised manuscript.

Lines 92/93 transpose “critical patient's condition" to patient's critical condition.

Answer: As required, we transposed “critical patient's condition” to “patient's critical condition”. See page 8, line 92 of the revised manuscript.

Line 97 change "being" to when.

Answer: As required, we changed “being” to “when”. See page 9, line 103 of the revised manuscript.

Reviewer 2 Report

This is an interesting paper describing the occurrence of candidemia due to C. glabrate in a severely ill diabetic patient suffering from COVID-19. The paper is generally well-written and requires more refinement to reach a balanced focus on the topic of candidemia.

1. In introduction authors clearly highlighted the COVID-19 pandemic and also how diabetes renders the inflicted patients to acquire (secondary) infection/ s. Indeed, numerous studies have shown the involvement of IPA among severely ill COVID-19 patients (DOI: 10.3390/jof6020091), while such a phenomenon seems underestimated in the context of candidiasis and/ or candidemia. Therefore, authors are encouraged to highlight this introduction. Thereafter, authors are encouraged to mention that C. glabrata is commensal, which lives on mucosal surfaces. On the other hand, the epidemiology of candidemia due to C. glabrata should be mentioned to highlight the importance of C. glabrata (DOI: 10.3390/jof6030138). Candida glabrata is the second leading cause of candidemia in some European countries (doi.org/10.1093/ofid/ofy358), USA (doi.org/10.1093/ofid/ofy358), and some Asian countries (DOI: 10.1093/mmy/myaa059), which is also amongst the most important MDR Candida species with a high level of tolerance against echinocandins and reduced susceptibility to azoles (DOI: 10.3390/jof6030138). This species also has a tropism to cause candidemia among the elderlies, therefore COVID-19 patients suffering from ARDS, who are mostly elderly, could be a favorite target of candidemia due to C. glabrata. This will also reveal the tragedy of echinocandin resistance in COVID-19 cases suffering from candidemia due to C. glabrata since this species has an intrinsically high MIC value against azoles and AMB is not well-tolerated among elderlies (also COVID-19 itself is associated with kidney injury which may further hamper the utility of AMB in this context).
2. The authors are encouraged to clarify the reference for the primers used to amplify and sequence the hotspot regions.
3. MALDI-TOF MS is not regarded as a resolutive tool to assess the genotypic relationship for Candida species isolates. Therefore, authors are encouraged to at least use an MLST scheme to show that isolate 1 was the parental isolate from which the resistant isolate was generated.
4. Lines 72-93 of discussion, please note and discuss that mutation in HS regions is not always associated with echinocandin resistant and some echinocandin-susceptible Candida glabrata isolates have been shown to have HS mutation and the infected patients showed therapeutic failure following echinocandin treatment (DOI: 10.11/myc.13104). Moreover, in some cases, echinocandin-resistant isolates may harbor HS mutation outside of HS region (DOI: 10.1080/22221751.2019.1684209 and DOI: 10.3390/jof6030138). All these clearly necessitate the combination of HS sequencing and echinocandin susceptibility testing as the most sensitive strategy to ensure choosing an appropriate antifungal therapy in the clinic.
5. Although authors discussed the rationale behind the case fatality, they did not show other risk factors known to be associated with candidemia for their cases, such as extensive use of antibiotics and multiple bacteremias.
6. Lines 68-71 of discussion. Please note that this study, ref #40, showed that caspofungin was associated with the highest rate of spontaneous mutation in HS regions when compared to anidulafungin and micafungin. Therefore, the use of caspofungin in this case as a strong mutation inducer in HS, may has resulted in the rapid development of echinocandin resistance and subsequent therapeutic failure.
7. The fatality of the case is a matter of debate and authors are encouraged to shorten this section, since the likelihood of death due to Candida glabrata is low considering several underlying conditions, multiple bacteremias, COVID-19, etc.

Author Response

This is an interesting paper describing the occurrence of candidemia due to C. glabrata in a severely ill diabetic patient suffering from COVID-19. The paper is generally well written and requires more refinement to reach a balanced focus on the topic of candidemia.

1. In introduction, authors clearly highlighted the COVID-19 pandemic and how diabetes renders the inflicted patients to acquire (secondary) infection/s. Indeed, numerous studies have shown the involvement of IPA among severely ill COVID-19 patients (DOI: 10.3390/jof6020091), while such a phenomenon seems underestimated in the context of candidiasis and/or candidemia. Therefore, authors are encouraged to highlight this introduction. Thereafter, authors are encouraged to mention that C. glabrata is commensal, which lives on mucosal surfaces. On the other hand, the epidemiology of candidemia due to C. glabrata should be mentioned to highlight the importance of C. glabrata (DOI: 10.3390/jof6030138). Candida glabrata is the second leading cause of candidemia in some European countries (DOI: 10.1093/ofid/ofy358), USA (DOI: 10.1093/ofid/ofy358), and some Asian countries (DOI: 10.1093/mmy/myaa059), which is also amongst the most important MDR Candida species with a high level of tolerance against echinocandins and reduced susceptibility to azoles (DOI: 10.3390/jof6030138). This species also has a tropism to cause candidemia among the elderlies, therefore COVID-19 patients suffering from ARDS, who are mostly elderly, could be a favorite target of candidemia due to C. glabrata. This will also reveal the tragedy of echinocandin resistance in COVID-19 cases suffering from candidemia due to C. glabrata since this species has an intrinsically high MIC value against azoles and AMB is not well-tolerated among elderlies (also COVID-19 itself is associated with kidney injury which may further hamper the utility of AMB in this context).

Answer: As suggested, we expanded the Introduction by adding a new paragraph to emphasize the importance of Candida glabrata and the issues related to secondary infections in COVID-19 patients, who may be at increasing risk of candidemia due to a Candida species difficult to treat with commonly used antifungal agents. As it can see, we added all the references suggested by the reviewer to support statements. See page 2, lines 44 to 58 of the revised manuscript.

2. The authors are encouraged to clarify the reference for the primers used to amplify and sequence the hotspot regions.

Answer: We added the reference for the primers used to amplify and sequence the hotspot regions. See page 3, line 41 of the revised manuscript.

3. MALDI-TOF MS is not regarded as a resolutive tool to assess the genotypic relationship for Candida species isolates. Therefore, authors are encouraged to at least use an MLST scheme to show that isolate 1 was the parental isolate from which the resistant isolate was generated.

Answer: We agree with the reviewer that MALDI-TOF MS may have a lower discriminative power than the methods currently used to assess the genotypic relationship among isolates from Candida species. Consistently, we added information about an MLST scheme to show that isolates 1 and 2 shared the same allelic profile. See page 4, lines 154 to 157 of the revised manuscript.

4. Lines 72-93 of discussion, please note and discuss that mutation in HS regions is not always associated with echinocandin resistant and some echinocandin-susceptible Candida glabrata isolates have been shown to have HS mutation and the infected patients showed therapeutic failure following echinocandin treatment (DOI: 10.1111/myc.13104). Moreover, in some cases, echinocandin-resistant isolates may harbor HS mutation outside of HS region (DOI: 10.1080/22221751.2019.1684209 and DOI: 10.3390/jof6030138). All these clearly necessitate the combination of HS sequencing and echinocandin susceptibility testing as the most sensitive strategy to ensure choosing an appropriate antifungal therapy in the clinic.

Answer: We expanded the discussion at the indicated lines to add comments about the relevant issues raised by the reviewer. To support these comments, we added all the references suggested by the by the reviewer. See pages 8 and 9, lines 93 to 99 of the revised manuscript.

5. Although authors discussed the rationale behind the case fatality, they did not show other risk factors known to be associated with candidemia for their cases, such as extensive use of antibiotics and multiple bacteremias.

Answer: We added a sentence about extensive use of antibiotics and multiple bacteremias as risk factors for development of candidemia in our case. See page 7, lines 34 to 35 of the revised manuscript.

6. Lines 68-71 of discussion. Please note that this study, ref #40, showed that caspofungin was associated with the highest rate of spontaneous mutation in HS regions when compared to anidulafungin and micafungin. Therefore, the use of caspofungin in this case as a strong mutation inducer in HS, may has resulted in the rapid development of echinocandin resistance and subsequent therapeutic failure.

Answer: We expanded the discussion at indicated lines taking into account the reviewer’s comments. See page 8, lines 67 to 70 of the revised manuscript.

7. The fatality of the case is a matter of debate and authors are encouraged to shorten this section, since the likelihood of death due to Candida glabrata is low considering several underlying conditions, multiple bacteremias, COVID-19, etc.

Answer: As suggested, we shortened the section regarding the fatality of the case. See page 7, lines 23 to 28 of the revised manuscript.