Cryptococcosis in Colombia: Analysis of Data from Laboratory-Based Surveillance 2017–2024

Round 1

Reviewer 1 Report

Overall, this is a timely, interesting and well-written study updating periodic published analyses containing multiple datasets on aspects of cryptococcosis - from different sources including hospitals, laboratories and other centres in Colombia. Its importance is in showing trends in incidence, epidemiology, laboratory parameters (in particular, including MICs), clinical and radiological features and outcomes- with implications for updated guidance on diagnosis, management etc - and underlining the importance of implementing national surveillance programs. Limitations of the study are addressed, including potential missed cases due to non-mandatory reporting and the lack of medium to longer term outcome data (especially with the high proportion of Non-WT MICs found with certain azole drugs).

1. "Diabetes" and "cirrhosis" are referred to as risk factors throughout the manuscript. Certainly, poorly controlled diabetes and decompensated cirrhosis are unequivocal risk factors. It would be helpful to know whether "diabetes" and "cirrhosis" were defined in this way in the data collection sheets (or not). If not, it would be worth considering such a subdivision in future.
2. Lines 79-81. I suggest that the authors specify the fact that more than two disease sites can include CNS infection, despite its own separate category, assuming that this is the case. Also, that the definition of relapse required a positive culture (ie IRIS was not the cause of an apparent relapse).
3. Para starting Line 96. Please confirm that all MICs were done in a Central laboratory or if not, that a Quality Control process operated to validate consistent results were obtained between laboratories.
4. Fig 1 and related text/tables: Is there/could there be a climatic or ecological component to the geographic differences?
5. Minor points in relation to the more genetically heterogenous types seen amongst C gattii cases (despite small numbers). Does the strong association of VGII with pulmonary disease, as observed in British Columbia, Canada, for example, hold true in Colombia? The higher proportion of non-WT MICs and differences between azoles is concerning, as noted by the authors. Again, despite small numbers, there are data from other countries on VG genotype-dependent differences in MIC but the clinical implications are uncertain. Analysis of the cases in this study, even though small in number, would add to the global data base. Will future studies by this group enable follow up of outcomes and help clarify triggers for changing antifungal drugs or therapeutic guidelines? Based on the results presented, the subject of future research needed in cryptococcosis could also be listed in a recommendations section at the end of the manuscript.
6. The non-WT MICs amongst C neoformans for flu, vori and posa are concerning and future studies that link them with clinical outcomes will be valuable.
7. Imaging studies. Minor point. CT and MRI data both include cerebral atrophy. How do the authors interpret this finding in the context of cerebral cryptococcosis as it is not usually considered as feature of cerebral cryptococcosis.

 

Author Response

Overall, this is a timely, interesting and well-written study updating periodic published analyses containing multiple datasets on aspects of cryptococcosis - from different sources including hospitals, laboratories and other centres in Colombia.

 

Its importance is in showing trends in incidence, epidemiology, laboratory parameters (in particular, including MICs), clinical and radiological features and outcomes- with implications for updated guidance on diagnosis, management etc - and underlining the importance of implementing national surveillance programs. 

 

Limitations of the study are addressed, including potential missed cases due to non-mandatory reporting and the lack of medium to longer term outcome data (especially with the high proportion of non-WT MICs found with certain azole drugs).

We thank the reviewer for the comments to the manuscript; as it was well understood, this is a passive and voluntary laboratory-based surveillance that we have done for many years, and that has allowed us to have a wide vision about Cryptococcosis in Colombia, although limitations that are highlighted in the text.

1. "Diabetes" and "cirrhosis" are referred to as risk factors throughout the manuscript. Certainly, poorly controlled diabetes and decompensated cirrhosis are unequivocal risk factors. It would be helpful to know whether "diabetes" and "cirrhosis" were defined in this way in the data collection sheets (or not). If not, it would be worth considering such a subdivision in future.

   Thank you for the comment.

   Indeed, in the survey (Table S2) diabetes and cirrhosis are listed as risk factors, but data of degree of compensation of the disease are not listed. We will consider the observation to include the suggested subdivision in the national survey. 
2. Lines 79-81. I suggest that the authors specify the fact that more than two disease sites can include CNS infection, despite its own separate category, assuming that this is the case. Also, that the definition of relapse required a positive culture (ie IRIS was not the cause of an apparent relapse).

   Thank you for the observations

   To clarify the concept, we modified the sentence as highlighted in line 89, page 3

   Line 89. Disseminated cryptococcosis was defined by a positive culture from at least two different sites, including CNS, or a positive blood culture

   • About relapses, we corrected the paragraph

    

   Line 90. Relapses were considered when a patient presented with a new clinical episode of cryptococcosis six or more months after the initial diagnosis, with a positive culture.
3. Para starting Line 96. Please confirm that all MICs were done in a Central laboratory or if not, that a Quality Control process operated to validate consistent results were obtained between laboratories

We confirm that determination of MICs were done in the National Reference Laboratory of the INS in Colombia. Additionally, INS participates in an external quality control for Antifungal Susceptibility Testing, sent by Malbran Institute of Argentina.

4. Fig 1 and related text/tables: Is there/could there be a climatic or ecological component to the geographic differences?

Could be, but we did not determine these differences in our surveillance

5. Minor points in relation to the more genetically heterogenous types seen amongst C gattii cases (despite small numbers).

• Does the strong association of VGII with pulmonary disease, as observed in British Columbia, Canada, for example, hold true in Colombia?

We did not do an analysis between association of pulmonary disease and VGII pattern. However, this relation may exist. Pulmonary disease was reported in 40 (87%) of patients with cryptococcosis caused by C. neoformans, whilst in only 3 patients (6.5%) with this clinical presentation, C. gattii was recovered (2 of these being VII pattern). In Table 3 we can observe that pulmonary form is more frequent in HIV negative patients (1.8 % in HIV + vs. 10.9% in HIV negative, p<0.0001), and C. gattii was recovered with higher frequency in HIV negative patients (2.9% HIV + vs. 13.8% HIV negative, p<0.0001).

• The higher proportion of non-WT MICs and differences between azoles is concerning, as noted by the authors.

We thank the comment, and we will be playing close attention to keep monitoring the antifungal susceptibility to the azoles in all the isolates received at the National reference laboratory as part of the surveillance.

• Again, despite small numbers, there are data from other countries on VG genotype-dependent differences in MIC but the clinical implications are uncertain.

We thank the comment, when analyzing our data, no genotype-dependent differences in MIC were seen, as NWT isolates for azoles remain in a high percentage. The following sentence was added:

In general, there is no genotype-dependent differences in MIC values for C. gattii isolates.

• Analysis of the cases in this study, even though small in number, would add to the global data base.

Thank you. We agree with the comment; publication will be a way of disseminating the findings.

• Will future studies by this group enable follow up of outcomes and help clarify triggers for changing antifungal drugs or therapeutic guidelines?

It is a very interesting but difficult task to carry out in Colombia. Once again we should remember that this was not a compulsory notification disease in our country for the reported period. We try to follow patients in the past with very low success, obtaining data from one or two centers.

• Based on the results presented, the subject of future research needed in cryptococcosis could also be listed in a recommendations section at the end of the manuscript.

Thank you for the comment.

Attending the suggestion, we emphasized at the end of the manuscript, based on our findings, the following:

Future work in Colombia should be focused on strengthening the surveillance with emphasis in the complete filling of the survey and submission of isolates to the National reference laboratory of the INS; include the mandatory determination of MICs and molecular pattern of the received isolates by the National Reference Center at the INS; and propose a patient tracking system in participating centers that report the highest number of isolates, highlighting the need of determining the MICs of the isolates. 

• The non-WT MICs amongst C neoformans for flu, vori and posa are concerning and future studies that link them with clinical outcomes will be valuable.

Once again we thank the suggestion and we will communicate our findings to the professionals of the centers that provide the higher number of surveys to determine what type of study we could perform. 

• Imaging studies. Minor point. CT and MRI data both include cerebral atrophy. How do the authors interpret this finding in the context of cerebral cryptococcosis, as it is not usually considered as feature of cerebral cryptococcosis?

We agree with the reviewer´s comment; cerebral cryptococcosis does not cause atrophy. However, cerebral atrophy is a frequent finding in patients that live with HIV in AIDS phase, which are an important number in our surveillance.

Reviewer 2 Report

Thank you for the opportunity to review this work. The authors conducted a retrospective analysis of cryptococcosis cases across Colombia from 2017–2024. My main comments are about the data source and analysis.

Major comments

• Introduction (lines 48–49): Please elaborate on the differences in ecological niches between neoformans and C. gattii.
• Incidence calculation: For the calculation of incidence rates, please clarify whether all included cases represent new cases each year. If the same patient was reported in multiple years, these data would more accurately reflect prevalence rather than incidence.
• Immunosuppressive therapy: If possible, please specify the corticosteroids or other immunosuppressive agents used, as these vary in potency and mechanisms of action.
• HIV-related data: What were the CD4 cell counts among patients with HIV? How many met criteria for AIDS?
• Diabetes-related data: What were the HbA1c levels in patients with diabetes?
• Section 3.4 (risk factors): These risk factors could be analyzed further using risk ratios. Although the authors state that HIV infection is the most important risk factor, simple percentage-based comparisons may be misleading due to differences in sample size. The risk factors listed in Table 1 should be analyzed using risk ratios with corresponding p-values.
• Clinical stratification: Analyses of symptoms/signs and treatment could be further strengthened by stratifying them according to clinical presentation.

Minor comments

• Methods: Please define “CSF” at its first mention.
• Figure 1: The information in Figure 1 is difficult to interpret due to poor resolution.

Author Response

1. Introduction (lines 48–49): Please elaborate on the differences in ecological niches between neoformansand  gattii.

Thank you for your comment. We included the following paragraph

On the other hand, C. neoformans and C. gattii have different ecological niches [7] and present differences in epidemiology and clinical manifestations [8,9]. C. neoformans and C. gattii are distributed across different ecological niches. C. neoformans is ubiquitous and frequently found in pigeon excreta and decaying wood worldwide, while C. gattii is more prevalent in tropical and subtropical regions and is strongly associated with eucalyptus and other trees, although its range is expanding into temperate zones [7].

2. Incidence calculation: For the calculation of incidence rates, please clarify whether all included cases represent new cases each year. If the same patient was reported in multiple years, these data would more accurately reflect prevalence rather than incidence.

All included cases represent new cases each year, therefore, we are presenting incidence rates, rather than prevalence.

3. Immunosuppressive therapy: If possible, please specify the corticosteroids or other immunosuppressive agents used, as these vary in potency and mechanisms of action.

Thank you for your comment. The survey points out the risk factors and no discrimination is made between the types of immunosuppressive agent used. Please refer to Table S2 

4. HIV-related data: What were the CD4 cell counts among patients with HIV? How many met criteria for AIDS?

Although CD4 cell count data is requested to be filled out in the survey (Table S2), only 42.6% register this information. Median of CD4 count for registered patients was 34. The following sentence was added:

CD4 cell count was registered in 42.6% of patients, with a median of 34 cells/mm³.

Every patient living with HIV and cryptococcosis was considered as being a patient with AIDS according to the CDC guidelines (https://www.cdc.gov/mmwr/preview/mmwrhtml/rr6303a1.htm)

5. Diabetes-related data: What were the HbA1c levels in patients with diabetes?

Thank you for your comment. However, in the survey (Table S2), there is no specific information about diabetes; therefore, this data is not available. 

6. Section 3.4 (risk factors): These risk factors could be analyzed further using risk ratios. Although the authors state that HIV infection is the most important risk factor, simple percentage-based comparisons may be misleading due to differences in sample size. The risk factors listed in Table 1 should be analyzed using risk ratios with corresponding p-values.

It is not possible to determine risk ratios as suggested since a same patient may have more than one risk factor as described in Table 1.

On the other hand, in Table 3 we did analyze risk factors between HIV positive and negative patients with the corresponding p-values.

The following sentence was added to the text:

Although HIV infection is the primary risk factor for cryptococcosis in our population (63%); furthermore, in Colombia, HIV infection is 157 times more frequent in patients with cryptococcosis.

7. Clinical stratification: analyses of symptoms/signs and treatment could be further strengthened by stratifying them according to clinical presentation.

Because most patients had neurocryptococcosis, data analysis presented in this study emphasizes its clinical manifestations. Other symptoms and signs related to other clinical presentations, such as pulmonary cryptococcosis, are not covered in the document.

Minor comments

• Methods: Please define “CSF” at its first mention

Thank you, it was defined as suggested

• Figure 1: The information in Figure 1 is difficult to interpret due to poor resolution.

Size of figure was enlarged in the main document. However, as requested by the journal author instructions, Figure 1 is uploaded as a separate file with the proper resolution.

Reviewer 3 Report

Dear authors:

I consider this is a good article that continue showing the epidemiological evolution of Cryptococcosis in Colombia.

There are only few points to clarify or correct:

1. Line 45. Correct "in several recent studies2
2. Line 78. Do you consider that Film array in CSF is reliable enough to diagnose cryptococcosis?
3. Line 115. Did you consider that Amphotericin B MIC value corresponds to the concentration that produced ≥ 50%  growth inhibition like azoles?
4. Table 1. What do you mean with "nodal clinical presentation?
5. Table 1: Do you consider cutaneous presentation as a primary cutaneous lesion?
6. Line 188. Please clarify the FA meaning
7. Table 2. Were there not LFA in serum or in CSF used to  diagnose cryptococcosis.
8. Line 450. Correct with
9. Line 460. Please correct "suspected" (it says suspicion)

 

 

 

 

Author Response

Major comments

Dear authors:

I consider this is a good article that continue showing the epidemiological evolution of Cryptococcosis in Colombia.

Dear reviewer, thank you for your concept about our paper.

 

1. Line 45. Correct "in several recent studies2

Thank you for the observation. The paragraph was modified

Meningeal cryptococcosis remains the most common etiology of meningitis in adults living with HIV in sub-Saharan Africa and an annual incidence of 152,000 cases of meningeal cryptococcosis resulting in 112,000 deaths has been estimated in several recent studies [2]

2. Line 78. Do you consider that Film array in CSF is reliable enough to diagnose cryptococcosis?

Yes, we consider that film array is reliable, in the clinical context of the patient.

3. Line 115. Did you consider that Amphotericin B MIC value corresponds to the concentration that produced ≥ 50% growth inhibition like azoles?

Thank you for the comment. The text was adjusted as follows:

AMB MIC readings were done where the lowest antifungal agent concentration prevented any discernable growth:

MICs of antifungals, except AMB, were the lowest drug concentrations that produced ≥50% growth inhibition compared to the growth control; for AMB, MIC is considered as the lowest antifungal agent concentration that prevents any discernable growth [17,18].

4. Table 1. What do you mean with "nodal clinical presentation?

Thank you for the observation: In the table legend we changed “nodal” by Lymph node”

5. Table 1: Do you consider cutaneous presentation as a primary cutaneous lesion?

No, cutaneous presentation was considered as secondary to a hematogenous dissemination 

6. Line 188. Please clarify the FA meaning

Thank you for the observation, FA is for Film Array.

• Overall, diagnosis of cryptococcosis was made by culture, direct examination, antigenemia and Film array.
• We also corrected Table 2 legend

  ** Diagnosis by Film array only in 5 patients

  We corrected Table S3

7. Table 2. Were there not LFA in serum or in CSF used to diagnose cryptococcosis.

In Table S3 are recorded the results of LFA that were very few; sample used was serum or CSF

8. Line 450. Correct with

Thank you for the suggestion; however, we did not find the mistake 

9. Line 460. Please correct "suspected" (it says suspicion)

Thank you for the observation. Words were re written as highlighted in the text:

Overall, according to the Global guideline for the diagnosis a management of cryptococcosis published in 2024, recommendation for diagnosis in patients with suspected or confirmed cryptococcosis require medical review for central nervous system, pulmonary or any other body site involvement, which include lumbar puncture with measurements of CSF opening pressure, glucose, protein and cell counts, microscopy, culture and CrAg quantification; tittering of blood CrAg, and culture of blood, sputum (or other respiratory specimens) and/or of other affected body sites; and brain and chest imaging [55].