Prevalence of Cryptococcal Antigenemia and associated factors among HIV/AIDS patients on second-line antiretroviral therapy at two hospitals in Western Oromia, Ethiopia.

BACKGROUND: Cryptococcosis is a global public health important infectious disease. HIV infection is the main risk factor estimated to account for 95% of cases in the middle- and low-income countries and 80% of the cases in high-income countries.
OBJECTIVE: The main aim of the study was to determine the prevalence and associated risk factors of Cryptococcal antigenemia (CrAg) among HIV/AIDS Patients on second-line ART Therapy at Ambo General Hospital and Nekemte Referral Hospital, Western Oromia, Ethiopia.
MATERIALS AND METHODS: Hospital-based cross-sectional study was employed from September 1, 2017, to October 30, 2017. Whole blood was tested for CrAg using Cryptococcal lateral flow assay (Immuno-Mycologics, Norman, OK, USA) according to the manufacturer's instructions. The collected data were analyzed using SPSS version 20 software. Binary logistic regression models were applied to assess the association between predictors and outcome variables at 95% CI. RESULT: Among the study participants, 115(62.8%) were females and the median age of the participants was 35 (IQR: 14) years. Majority, 169(92.3%), have been living with HIV for ≥ 5.6 years and 124 (67.8%) stayed on 2nd line ART for an average of 2.5 years. The overall prevalence of Cryptococcal antigenemia in the study participants was 7.7% (14/183). Being male [AOR, 95% CI: 4.78(1.14, 20.1)], poor adherence to ART [AOR, 95% CI: 0.12(0.03, 0.4)], occupational exposures to contaminated soil [AOR, 95% CI: 6.81(1.38, 33.4)], having non-separated house from chickens [AOR, 95% CI: 0.06(0.01, 0.51)], CD4 T+ cell levels ≤ 100 cell/μL [AOR, 95% CI: 6.57(1.9, 23.3)] and viral load >1000 copies/mL [AOR, 95% CI: 11.7(2.4, 57.8)] were significant predictors of Cryptococcal antigenemia (P≤ 0.05).
CONCLUSION: The prevalence of Cryptococcal Antigenemia was significantly high in this study. Being male, occupational exposure to contaminated soil with avian droppings, CD4+ T cell levels <100 cell/μL and viral load >1000 copies/mL were significant predictors of Cryptococcal antigenemia. Therefore, public health measures, adherence to ART and early treatment are recommended.

Introduction

Cryptococcosis is a zoonotic disease and it is one of the life-threatening opportunistic mycoses in HIV/AIDS patients. The disease is caused by a capsulated yeast of the Cryptococcus neoformans complex [1]. The yeast is found in the environment mostly associated with pigeon droppings, soil and avian excreta throughout the world [2]. The yeast mainly causes pulmonary infection upon inhalation of the spores and symptoms are pneumonia-like. In immunosuppressed patients, the fungi disseminate to other sites commonly to the meninges and cause life-threatening Cryptococcal meningitis [3].

Cryptococcosis is the commonest fungal disease in immunocompromised individuals and it infects more than one-third (60–70%) of HIV/AIDS patients [4, 5]. The epidemiology of crptococcosis has been increased in the past three decades governed by the progress of the HIV pandemic. Although a combination of treatments (ART and anti-fungal drugs) has been contributing to the dropping of mortality in high income countries (HICs), the death rate in middle and low income countries (MLICs) is still high. Specifically, CNS cryptococcosis is deadly unless treated [4].

Detecting the presence of cryptococcal antigens in the patients’ blood, Cryptococcal antigenemia (CrAg) tests, is the commonest method for diagnosing Cryptococcal meningitis (CM). The CrAg test can detect the target antigen from peripheral blood on average of 22 days prior to the development of CM and it is estimated that about 11% of patients will have positive CrAg test more than 100 days prior to the onset of the disease, CM [5].

The survival rate and the quality of life of HIV/AIDS patients with cryptococcosis have been greatly improved after the administration of a combination of antiretroviral and antifungal therapy [6,7]. However, HIV/AIDS patients with CD4+ T cell counts <100 cells/μL are at risk of Cryptococcosis and the situation is worse in those who are not taking ART. Therefore, early screening and treatment of HIV/AIDS patients for cryptococcosis is of a vital significance [8].

Although previous studies in Ethiopia reported about the prevalence of cryptococcal antigenemia in HIV/AIDS patients, there is a paucity of published data regarding cryptococcosis among HIV/AIDS patients under second-line ART specifically in the study area. This study is, therefore, aimed to determine the prevalence and associated risk factors of Cryptococcal antigenemia among HIV/AIDS patients on 2nd line ART at Ambo General Hospital and Nekemte Referral Hospital of Western Oromia, Ethiopia. We believe this is the first study in the specific study area and will contribute to the betterment of prevention and treatment activities of crptococcosis in Ethiopia

Materials and methods

Study setting and context

A hospital-based cross-sectional was conducted at Ambo General Hospital and Nekemte Referral Hospital, Western Oromia, Ethiopia, from September 1, 2017, to October 30, 2017. Ambo and Nekemte towns are located at 115 and 327 kilometers West of the Capital Addis Ababa, respectively. These two study areas were selected by lottery method from eight western Oromia hospitals which serve for a significant number of HIV/AIDS patients on second-line ART. Adult HIV/AIDS patients on second-line ART regimens were 129 and 154 at Ambo General Hospital and Nekemte Referral Hospital, respectively.

Study population

The study population for this study was all consented HIV/AIDS patients on 2nd line ART at Ambo General Hospital and Nekemte Referral Hospital. All HIV/AIDS patients under second-line anti-retroviral treatment aged ≥18-years-old who didn’t take anti-cryptococcosis drugs in the past three months were included in the study.

Sample size determination and sampling technique

The sample size was calculated using a single population proportion formula taking 4.1% prevalence of Cryptococcal Antigenemia from previous study [9], a margin of error 5% and 95% confidence level.

n = (Zα/2)2 p (1-p)/d2

Zα/2 = standard normal variable at 95% confidence level (1.96).

d = precision (tolerable margin of error)

n = Zα/2 p (1-p)/w2 = (1.96)2 x 0.041 (1–0.041)/(.05)2 = 60 patients

Considering 15% as non-response rate, the final sample size is: (60 * 0.15) + 60 = 69 participants.

By using Design Effect = 2.7

n = 69 * 2.7 = 186 participants.

The study participants were then selected from the two hospitals using proportional cluster sampling technique as follow:

n is a sample size of the ihospital

Ni is a total of HIV-infected patients on second-line ART registered for ART in ihospital.

n = n1 + n2is the total sample size, which is 186

N = N1 + N2 is the total number of HIV-infected patients on second-line ART registered for ARTin the two hospitals.

Finally, a total of 186 (101 from Nekemte Referral Hospital and 85 from Ambo General Hospital) study participants were consecutively enrolled.

Data collection

Site assessment and pretest was done prior to the actual data collection in an unselected hospital using 5% of sample size and data were collected using structured questionnaires. The questionnaire was developed in English and translated into the local language; Afan Oromo. Data were collected by trained nurses and medical laboratory technologists. Collected questionnaires were cross-checked daily for completeness.

Laboratory procedures

Whole blood was collected from each study participants. CrAg Lateral Flow Assay (CrAg LFA) and CD4+ T-cell counts were done following standard protocols and manufacturer’s instructions [10,11]. All CrAg LFA tests were done in duplicate by two laboratory technologists to minimize bias and avoid errors. Briefly, one drop of the patient specimen is added to the EDTA tube and then the CrAg LFA kit is inserted and incubated for 10 minutes. The test is interpreted “positive” if two lines appear on both the control and test lanes, “negative” if one line appears on only the control line and “invalid” if a line appears only on the test lane.

The number of viral RNA copies (viral load) is determined for every HIV/AIDS patient in Ethiopia using Real-Time Reverse transcription HIV-1 Polymerase Chain reaction (RT-PCR) (Abbott MolecμLar, Inc., Des Plaines, IL) at Ethiopian Public Health Institute and Nekemte Regional Laboratory [12] while CD4+ T cell count was done at the respective health facilities. Therefore, we didn’t collect additional blood for the purpose of reverse transcription polymerase chain reaction (RT-PCR) and CD4+ T cell count in our study rather we used laboratory data which were done simultaneously with the CrAg test for the purpose of patient follow-up.

Data quality assurance

The questionnaires were carefully designed, pre-coded, and then pretested to minimize errors. Data collectors and data assistants were trained prior to data collection. The collected data were checked daily for completeness and callbacks were arranged for any incomplete data. Experienced laboratory personnel was recruited to perform tests using appropriate laboratory facilities which have a separate area for sample preparation and analysis and access to water of appropriate quality. Adequate blood specimens were collected using carefully labeled dry EDTA tubes. The specimens were kept free of contamination from water and soil. All CrAg, CD4+ T cell count and viral load test results were encoded and reported appropriately. All CrAg tests were run together with positive and negative control samples. All reagents were checked for expiry date and all procedures were done following the manufacturer's guidelines and standard operating procedures.

Data analysis

Cleaned and coded data were entered into EPI-data version 3.1 software and exported to SPSS version 20 software for analysis. Descriptive data were presented using frequency statistics. Binary logistic regression models were used for inferential statistics to identify independent predictors of Cryptococcal antigenemia at 95% CI.

Ethics approval considerations

The study was conducted after it was ethically reviewed and approved by the Institutional review board (IRB) of the research directorate of Wollega University. Then a letter informing the respective hospitals was written from Wollega University and permission obtained. All the information obtained from the study participants was coded to maintain confidentiality. All participants were adults to provide informed consent in line with local laws. The IRB approved the use of oral consent documented by a witness after the objectives of the study had been explained. The positive results were timely reported to the clinicians for appropriate intervention.

Results

Socio-demographic characteristics of study participants

A total of 183 HIV-infected patients on second line ART were enrolled in the study with a 98% response rate. Majority of the participants were females 115(62.8%) and the median age of the study participants was 35 (IQR: 14) years. About 81.4% of them had monthly income <1500 birr, 132(72.1%) were urban residents and 97(53%) were married ().

Clinical characteristics of study participants

The mean duration of the patients living with HIV was 8.75 years. The mean duration of first-line ART was 4.5 years and 3.7 years for second-line ART. The mean baseline (before starting treatment) CD4+ T cell level of the participants was 163.2±82 cells/μL. The mean current CD4+ T cells count was 434.4±286.3 cells/μL whereas the median viral load was 10110 HIV RNA copies/ml ().

Prevalence of Cryptococcal Antigenemia

The overall prevalence of Cryptococcal Antigenemia was 7.7% (14/183) of which 71% (10/14) were males (). Among CrAg positive patients, about 86% (12/14) had viral load of >1000 copies/mL and about 71% (10/14) had baseline CD4+ T cell count <100 cells/μL ().

Assessment of associated risk factors for cryptococcosis

In binary logistic regression, age, marital status, education, occupation, income, residence, distance from a health facility, duration of living with HIV, duration with first-line ART & second-line ART and baseline CD4 count were not significantly associated with cryptococcal antigenemia (P > 0.05). Gender, patients' adherence to ART, occupational exposures to the soil, having separated house from domestic animals like hens, self-reported headache & weight loss, current CD4+ T cells, and viral load were associated with a higher prevalence of cryptococcal antigenemia in the crude analysis ().

After adjusting for confounding factors, cryptococcal antigenemia was significantly higher among males [AOR,95% CI: 4.8(1.14, 20), P <0.05], occupational exposure to soil [AOR, 95% CI: 6.8(1.38, 33), P < 0.05], non-separated house from domestic animals like hens [AOR, 95% CI: 0.06(0.01, 0.51), P < 0.05], poor adherence to ART [AOR, 95% CI: 0.12(0.03, 0.4), P< 0.05], CD4+ T cell counts ≤ 100 cell/μL [AOR, 95% CI: 6.57(1.9, 23.3), P < 0.05] and higher viral load (HIV RNA >1000 copies/mL) [AOR, 95% CI: 11.7(2.4, 57.8), P < 0.05]. Therefore, male HIV/AIDS patients, those occupationally exposed to soil, having house non-separated from domestic animals, poor adherence, low CD4+ T cell count and with high viral load were 4.8, 6.8, 0.06, 0.12, 6.57 and 11.7 times more likely to harbor cryptococcal antigenemia than their counters, respectively.

Discussion

In this study, the prevalence of Cryptococcosis among HIV/AIDS patients on second-line ART attending two Western Oromia hospitals was determined. The prevalence in the current study (7.7%) is comparable to a study conducted in Addis Ababa (8.4%) [13] and Indonesia (7.1%) [14]. But this result is higher than a study conducted in North West Ethiopia (0.5%) [15], South West London (5%) [16], Southern Nigeria (5.1%) [17] and Haiti (1.1%) [18]. However, it lower than the reported prevalence of 10.2% from South East Ethiopia [9], 33% from Kenya [19] and 8.9% from Nigeria [20]. These difference might be due to differences in the study population as the current study enrolled only HIV/AIDS patients under second-line ART, the geographical and weather differences as the transmission of Cryptococcus is geographically different, differences in laboratory protocols employed and differences in health care system outside Ethiopia.

Age, baseline CD4 count, duration of HIV infection and duration of ART didn’t significantly affect the prevalence of Cryptococcosis among HIV-infected patients on Second Line ART which is partly in agreement with a previous study and A higher rate of a headache in this study is comparable to other reports in Ethiopia [9]. Unlike previous studies [17, 19, 20], fever, headache and elder age were not significantly associated with an increased risk of Cryptococcal Antigenemia in the current study. This possible reason for these differences could be due to better management of the disease in elders, and better prevention of other diseases causing headache and fever.

The high positivity rate of Cryptococcus infection in this study implies the burden of Cryptococcal infection among HIV/AIDS patients on second-line ART is of public health importance. In this study, the distribution of CrAg among female and male HIV patients was different. Male patients had higher seropositivity (71.4%) compared to their female (28.6%) counterparts. In the United States, more men compared to women have been reported to carry a higher burden of cryptococcal infections [21]. The multivariate analysis revealed that there was a significantly higher prevalence of Cryptococcus infections in males than in females. This might be due to the fact that males have more outdoor activities in Ethiopia which could expose them to the droppings of avians and which in turn leads to Cryptococcal infection. In contrast to our findings, a study done in Nigeria [22] found out that female patients had a higher prevalence compared to male patients. This difference might be due to working habit and cultural differences between the two countries as males are mostly responsible for outdoor activities in Ethiopia.

The majority of the Cryptococcal Antigenemia positive cases (85.7%) were in the age group of 29–38 and 39–48 years in the current study; although, age was not a significant predictor of cryptococcosis. This finding is comparable to the study done in Nigeria [22]. Cryptococcal antigenemia was slightly higher in those with a shorter duration of second-line ART which means second-line ART is playing a critical role in preventing opportunistic infections.

The distribution of Cryptococcal Antigenemia among patients in this study was highly varying based on CD4+T cell counts as 50% of CrAg positive patients had CD4+ T cell counts below 100 cells/μL. Several studies done in different countries have also reported a consistently higher prevalence of Cryptococcal Antigenemia in patients with lower CD4+ T cell counts [23]. Our findings are comparable to other studies done in Uganda [24] and Cambodia [25] where authors reported a higher prevalence of cryptococcal antigenemia associated with low CD4+ T cell counts. The association between the two variables could be due to the fact that low CD4+ T cell counts predispose HIV-infected patients to opportunistic infections because of the immunosuppression. In resource-limited settings, screening of patients with a CD4+ T cell count less than 100 cells/μL for Cryptococcus infections may be more clinically relevant as CD4+ T cell tests are currently more accessible.

The prevalence of Cryptococcal Antigenemia was 14.28% in patients with a baseline CD4+T cell count of between 101–200 cells/μL. A recent study in Ethiopia reported an estimated serum Cryptococcal Antigenemia prevalence of 5.8% among HIV-infected adults with CD4+ T cell counts between 201–350 cells/μL [9]. Unlike this, the current study reported a 42.9% prevalence Cryptococcal Antigenemia in patients with current CD4+ T cell counts of >200 cells/μL but this higher prevalence of CrAg among patients with low CD4+ T cell counts is consistent with a study done in South Africa [26]. Differences in culture, lifestyle, using farming as a source of income and other related factors play a significant role for the observed differences across studies as the transmission of Cryptococcus neoformans is governed by all these factors.

In this study, high viral load was an independent predictor of cryptococcal antigenemia which is in line with a study from Kenya [27]. In general, being male, occupational exposures to the soil, having a non-separated house from domestic animals like hens and chicken, CD4+ T cell counts ≤100 cells/μL and viral load >1000 RNA copies/mL were independent predictors of Cryptococcal Antigenemia.

Although this study has limitations like small sample size, non-random sampling technique, and limited study area, it could play an important role in combating death secondary to cerebral meningitis due to Cryptococcal antigenemia among HIV/AIDS patients.

Conclusion

The prevalence of Cryptococcal Antigenemia in patients under second line ART is significantly high in the study area. Being male, poor adherence to ART, having a house not separated from domestic animals like hens, occupational exposures to contaminated soil with avian droppings, low CD4+ T cells count and high viral load was significant risk factors that increased cryptococcal antigenemia. Public health measures, early diagnosis, and treatment are vital to prevent Cryptococcal meningitis and death among HIV/AIDS patients. Further large-scale longitudinal studies are recommended to explore the risk factors and maximize the benefits of ART.

Questionnaires used to collect data from HIV/AIDS patients.

(DOCX)

Click here for additional data file.

27 Sep 2019

PONE-D-19-20216

Prevalence of Cryptococcal Antigenemia and Associated Factors Among HIV/AIDS Patients on Second-Line Antiretroviral Therapy at Two Hospitals in Western Oromia, Ethiopia

PLOS ONE

Dear Mr Mengist,

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.

REQUIRED: Clarify the CD4 count and the population being studied, as described in the reviewer comments.

REQUIRED: Address All reviewer comments individually in the "Response to Reviewers" document included with the resubmission.

We would appreciate receiving your revised manuscript by Nov 11 2019 11:59PM. When you are 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.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

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). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Kirsten Nielsen, Ph.D

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 http://www.journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and http://www.journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

Additional Editor Comments (if provided):

[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

**********

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

Reviewer #1: 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

**********

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

**********

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: Authorship order is different on the manuscript than in the PLoS One system.

Abstract Results: would prefer you to specify the median age (with interquartile range). % married is of no value and is distracting. Would remove that.

% living with HIV and/or on ART -consider changing units to years instead of months. This is more intuitive/understandable for readers

Introduction:

Line 59 -Define MLICs

Line 61 -CrAg isn't antibodies, it's antigen. would remove the term anti-cryptococcal antibodies. replace with "antigen", same with line 63

Methods:

Line 90 -don't use contractions in formal writing. Same with line 132.

Results:

Line 162: don't capitalize "second line ART"

Line 162 -saying 35% were in one age group is unhelpful. State the median age and interquartile range.

Line 168 -change to years

Line 170 -is this an error? the baseline CD4 was 163 +/- 182? That doesn't make sense.

Line 173 -the prevalence is 7.7%. is this among peoplew ith CD4<100? On line 170 it says mean CD4 was 434. Very confused about the population you're describing.

Line 221: Men tend to have more advanced HIV disease (ie. lower CD4 counts)

Line 229: It's not clear from your results what the CD4 counts were of your patient population and those who were CrAg+

Line 257: higher than what?

In your figure, label your Y axis. Don't use decimal places unless it's useful. And put percentages behind your numbers.

**********

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

[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 to be viewed.]

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 us at figures@plos.org. Please note that Supporting Information files do not need this step.

30 Sep 2019

Response to the reviewer

1. Authorship order is different on the manuscript than in the PLoS One system.

Response: We have communicated with the journal office for another paper and confirmed the authorship order in the manuscript is the final acceptable order. The journal has similar rules which we thought will be applicable for this manuscript, too. It was difficult for the corresponding author to put his name last in the editorial manager.

2. Abstract Results: would prefer you to specify the median age (with interquartile range). % married is of no value and is distracting. Would remove that.

% living with HIV and/or on ART -consider changing units to years instead of months.

Response: The age was described with median and IQR and we removed % marriage. And we changed unit of stay with HIV or ART in years instead of months.

3. Introduction: Line 59 -Define MLICs Line 61 -CrAg isn't antibodies, it's antigen. would remove the term anti-cryptococcal antibodies? replace with "antigen", same with line 63

Response: We defined MLICs (Middle and low income countries) and HICs (High income countries). We removed the term “anti-cryptoccocal antibodies” and changed the “antibody” miswording to “antigen”.

4. Methods:

Line 90 -don't use contractions in formal writing. Same with line 132.

Response: We fixed the abovementioned issue.

5. Results: Line 162: don't capitalize "second line ART"

Line 162 -saying 35% were in one age group is unhelpful. State the median age and interquartile range.

Response: We modified the phrase “Second Line ART” into “second line ART” and the median age was used than mean age.

6. Line 168 -change to years. Line 170 -is this an error? the baseline CD4 was 163 +/- 182? That doesn't make sense. Line 173 -the prevalence is 7.7%. is this among people with CD4<100? On line 170 it says mean CD4 was 434. Very confused about the population you're describing.

Response: We changed months to years in line 168. Line 170…it was error. Baseline CD4+ T cell count was 163 +/- 82, not 163 +/- 182. In line 173….the prevalence of CrAg test positivity was 7.7% from all study participants. But 50% prevalence was among participants whose current mean CD4+ T cell count <100 cells/µL. plus the phrase saying “….CD4 was 434….” is about the current mean CD4 T cell count. Thus our study population are those with current mean CD4 T cell count of 434, but their baseline (before starting treatment) was 163+/-82.

7. Line 221: Men tend to have more advanced HIV disease (ie. lower CD4 counts). Line 229: It's not clear from your results what the CD4 counts were of your patient population and those who were CrAg+

Response: Line 221…it doesn’t necessarily mean men has advanced disease than women rather men usually have outdoor activities in Ethiopia which exposes them for Cryptococcosis infection which in turn resulted higher prevalence of CrAg test positivity among men. Line 229……We included the proportion of CrAg positive with respect to CD+ T cell count in results section.

8. Line 257: higher than what?

Response: Line 260….higher. it is corrected into “….was significantly ‘high’ in the study area”.

9. In your figure, label your Y axis. Don't use decimal places unless it's useful. And put percentages behind your numbers.

Response: The figure was modified accordingly.

Submitted filename: Rebutal letter.docx

Click here for additional data file.

12 Nov 2019

Prevalence of Cryptococcal Antigenemia and Associated Factors Among HIV/AIDS Patients on Second-Line Antiretroviral Therapy at Two Hospitals in Western Oromia, Ethiopia

PONE-D-19-20216R1

Dear Dr. Mengist,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.

Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

With kind regards,

Kirsten Nielsen, Ph.D

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

22 Nov 2019

PONE-D-19-20216R1

Prevalence of Cryptococcal Antigenemia and Associated Factors Among HIV/AIDS Patients on Second-Line Antiretroviral Therapy at Two Hospitals in Western Oromia, Ethiopia

Dear Dr. Mengist:

I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

For any other questions or concerns, please email plosone@plos.org.

Thank you for submitting your work to PLOS ONE.

With kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Kirsten Nielsen

Academic Editor

PLOS ONE

Table 1

Socio-demographic characteristics of HIV-infected patients attending second-line ART at Ambo General Hospital and Nekemte Referral Hospital, Western Oromia, Ethiopia from September 1, 2017, to November 30, 2017.

Variables	Frequency	Percentages
Age (years)
18–28	52	28.4
29–38	64	35
39–48	46	25.1
49–58	16	8.7
59–68	3	1.6
> 68	2	1.1
Sex
Female	115	62.8
Male	68	37.2
Marital Status
Single	33	18
Married	97	53
Divorced	19	10.4
Widowed	29	15.8
Separated	5	2.7
Educational Status
Illiterate	51	27.9
Read & write	12	6.6
Primary School	64	35
Secondary School	43	23.5
College & above	13	7.1
Occupation
Employee	29	15.8
Farmers	37	20.2
Merchants	49	26.8
Students	21	11.5
Housewife	22	12
Daily Laborer	13	7.1
Others	12	6.6
Monthly Income
≤ 1500 birr	149	81.4
>1500 birr	34	18.6
Residence
Rural	51	27.9
Urban	132	72.1

Table 2

Clinical characteristics of HIV-infected patients attending Second-Line ART at Ambo General Hospital and Nekemte Referral Hospital; Western Oromia, Ethiopia; from Sep. 1, 2017, to Nov. 30, 2017.

Variables		Frequency	Percentages
Years lived with HIV	0.5–5.5	14	7.7
	≥5.5	169	92.3
Years on first line ART	<0.5	13	7.1
	0.5–5.5	107	58.5
	≥5.5	63	34.4
Years on second line ART	<0.5	21	11.5
	0.5–5.5	124	67.8
	≥5.5	38	20.8
Adherence to ART	Poor	39	21.3
	Good	144	78.7
The distance of the hospital	<1 Km	29	15.8
	1–3 Km	75	41
	>3 Km	79	43.2
Occupational Exposures to blood	Yes	4	2.2
	No	179	97.8
Occupational Exposures to soil	Yes	85	46.4
	No	98	53.6
Domestic animals like hen present	Yes	82	44.8
	No	101	55.2
House separated from the hen	No	15	18.3
	Yes	67	81.7
The patient was	Inpatient	8	4.4
	Outpatient	175	95.6
Fever	Present	45	24.6
	Absent	138	75.4
A headache	Present	93	50.8
	Absent	90	49.2
Skin Lesion	Present	32	17.5
	Absent	151	82.5
A cough	Present	43	23.5
	Absent	140	76.5
Neck Stiffness	Present	8	4.4
	Absent	175	95.6
Weight Loss	Present	38	20.8
	Absent	145	79.2
Night Sweatiness	Present	35	19.1
	Absent	148	80.9
Blurred Vision	Present	12	6.6
	Absent	171	93.4
Baseline CD4+ T counts (cells/μL)	≤100	85	46.4
	101–200	43	23.5
	>200	55	30.1
Current CD4+ T counts (cells/μL)	≤100	24	13.1
	101–200	19	10.4
	>200	140	76.5
HIV RNA Copies/ mL	≤1000	134	73.2
	>1000	49	26.8

Table 3

Cross-tabulation of CrAg among HIV-infected patients on 2nd line ART at Ambo General Hospital and Nekemte Referral hospitals, Western Oromia, Ethiopia from September 1 to November 30, 2017.

Variables		CrAg LF Test Result		Total
		Positive	Negative
Age (years)	18–28	0(0%)	52(100)	52(100%)
	29–38	5(8%)	59(92%)	64(100%)
	39–48	7(15.2)	39(84.8)	46(100%)
	49–58	2(12.5)	14(87.5)	16(100%)
	59–68	0(0%)	3(100%)	3(100%)
	>68	0(0%)	2(100%)	2(100%)
Sex	Female	4(3.5%)	111(96.5)	115(100%)
	Male	10(14.7)	58(58.3)	68(100%)
Marital Status	Single	3(9.1%)	30(90.9%)	33(100%)
	Married	6(6.2%)	91(93.8%)	97(100%)
	Divorced	3(15.8%)	16(84.2%)	19(100%)
	Widowed	2(6.9%)	27(93.1%)	29(100%)
	Separated	0(0%)	7(100%)	7(100%)
Educational Status	Illiterate	2(3.9%)	49(96.1%)	51(100%)
	Read & write	0(0%)	12(100%)	12(100%)
	Primary School	9(14.1%)	55(85.9%)	64(100%)
	Secondary School	1(2.3%)	42(97.7%)	43(100%)
	Above college	2(15.4%)	11(84.6%)	13(100%)
Occupation	Employee	4(13.8%)	25(86.2%)	29(100%)
	Farmers	4(10.8%)	33(89.2%)	37(100%)
	Merchants	1(2%)	48(98%)	49(100%)
	Students	0(0%)	21(100%)	21(100%)
	Housewife	0(0%)	22(100%)	22(100%)
	Daily Laborer	4(30.8%)	9(69.2%)	13(100%)
	Others	1(8.3%)	11(91.7%)	12(100%)
Income	≤1500	12(8.1%)	137(91.9%)	149(100%)
	>1500	2(5.9%)	32(94.1%)	34(100%)
Residence	Rural	4(7.8%)	47(92.2%)	51(100%)
	Urban	10(7.6%)	122(92.4)	132(100%)
Years since diagnosed positive for HIV	<0.5	2(14.3%)	12(85.7%)	14(100%)
	0.5–5.5	12(7.1%)	157(92.9%)	169(100%)
Years on first line ART	<0.5	0(0%)	13(100%)	13(100%)
	0.5–5.5	10(9.3%)	97(90.7%)	107(100%)
	≥5.5	4(6.3%)	59(93.7%)	63(100%)
Years on second line ART	<0.5	2(9.5%)	19(90.5%)	21(100%)
	0.5–5.5	10(8.1%)	114(91.9%)	124(100%)
	≥5.5	2(5.3%)	36(94.7%)	38(100%)
Adherence	Poor	10(25.6%)	29(74.4%)	39(100%)
	Good	4(2.8%)	140(97.2%)	144(100%)
The distance of the hospital	<1 Km	0(0%)	29(100%)	29(100%)
	1–3 Km	7(9.3%)	68(90.7%)	75(100%)
	>3 Km	7(8.9%)	72(91.1%)	79(100%)
Expose to blood	Yes	1(25%)	3(75%)	4(100%)
	No	13(7.3%)	166(92.7%)	179(100%)
Expose to soil	Yes	12(14.1%)	73(85.9%)	85(100%)
	No	2(2%)	96(98%)	98(100%)
House separated from the hen	No	7(43.8%)	9(56.2%)	16(100%)
	Yes	2(3%)	65(97%)	67(100%)
Patient was	Inpatient	1(12.5%)	7(87.5%)	8(100%)
	Outpatient	13(7.4%)	162(92.6%)	175(100%)
Fever	Present	3(6.7%)	42(93.3%)	45(100%)
	Absent	11(8%)	127(92%)	138(100%)
Headache	Present	12(12.9%)	81(87.1%)	93(100%)
	Absent	2(2.2%)	88(97.8%)	90(100%)
Skin Lesion	Present	5(15.6%)	27(84.4%)	32(100%)
	Absent	9(6%)	142(94%)	151(100%)
Cough	Present	4(9.3%)	39(90.7%)	43(100%)
	Absent	10(7.1%)	130(92.9%)	140(100%)
Neck Stiffness	Present	2(25%)	6(75%)	8(100%)
	Absent	12(6.9%)	163(93.1%)	175(100%)
Weight Loss	Present	6(15.8%)	32(84.2%)	38(100%)
	Absent	8(5.5%)	137(94.5%)	145(100%)
Night Sweat	Present	4(11.4%)	31(88.6%)	35(100%)
	Absent	10(6.8%)	138(93.2%)	148(100%)
Blurred Vision	Present	0(0%)	12(100%)	12(100%)
	Absent	14(8.2%)	157(91.8%)	171(100%)
Baseline CD4+ T cell count (Cells/μL)	≤100	10(11.8%)	75(88.2%)	85(100%)
	101–200	2(4.7%)	41(95.3%)	43(100%)
	>200	2(3.6%)	53(96.4%)	55(100%)
Current CD4+ T cell count (Cells/μL)	≤100	7(29.2%)	17(70.8%)	24(100%)
	101–200	1(5.3%)	18(94.7%)	19(100%)
	>200	6(4.3%)	134(95.7%)	140(100%)
HIV RNA Copies/mL	≤1000	2(1.5%)	132(98.5%)	134(100%)
	>1000	12(24.5%)	37(75.5%)	49(100%)

Table 4

Assessment of risk factors associated with CrAg among HIV patients on second line ART at two hospitals, Western Oromia, Ethiopia from September 1, 2017, to November 30, 2017.

Variables		CrAg LF Test Result		COR(95% CI)	P-value	AOR(95% CI)	P-value
		PositiveN (%)	NegativeN (%)
Sex	Female	4(3.5%)	111(96.5)	1		1
	Male	10(14.7)	58(58.3)	4.8(1.4–15.9)	0.011*	4.8(1.14–20)	0.032*
Adherence to ART	Poor	10(25.6%)	29(74.4%)	0.08(0.02–0.3)	0.000*	0.12(0.03–0.4)	0.001*
	Good	4(2.8%)	140(97.2%)	1		1
Exposure to soil	Yes	12(14.1%)	73(85.9%)	7.89(1.71–36)	0.008*	6.8(1.38–33)	0.018*
	No	2(2%)	96(98%)	1		1
Separated house from hen	No	7(43.8%)	9(56.2%)	0.04(0.01–0.2)	0.000*	0.06(0.01–0.5)	0.01*
	Yes	2(3%)	65(97%)	1		1
Headache	Present	12(12.9%)	81(87.1%)	1		1
	Absent	2(2.2%)	88(97.8%)	0.15(0.03–0.7)	0.016*	2.4(0.26–22.8)	0.4
Weight Loss	Present	6(15.8%)	32(84.2%)	1		1
	Absent	8(5.5%)	137(94.5%)	0.31(0.1–0.9)	0.042*	0.2(0.03–1.13)	0.06
Current CD4+ T cell count (Cells/μL)	≤100	7(29.2%)	17(70.8%)	9.2(2.76–30.6)	0.000*	6.57(1.9–23.3)	0.004*
	101–200	1(5.3%)	18(94.7%)	1.2(0.14–10.9)	0.84	0.8(0.09–7.6)	0.85
	>200	6(4.3%)	134(95.7%)	1		1
HIV RNA Copies/ml	≤1000	2(1.5%)	132(98.5%)	21.4(4.6–99.9)	0.000*	11.7(2.4–57.8)	0.002*
	>1000	12(24.5%)	37(75.5%)	1		1

*significance at P ≤ 0.05, AOR = adjusted odds ratio, COR = crude odds ratio