A Rapid Screening Program for Histoplasmosis, Tuberculosis, and Cryptococcosis Reduces Mortality in HIV Patients from Guatemala

Opportunistic infections (OIs) and advanced HIV disease (AHD) contribute to HIV-related mortality. Here, we analyzed the situation of AHD and OIs in a cohort of newly diagnosed HIV patients from Guatemala. We included 2127 adult patients from 13 facilities across the country during 2017 to 2018. Patients were screened for tuberculosis (TB), nontuberculous mycobacteria (NTM), histoplasmosis, and cryptococcal disease, independently of their CD4 cell count. Of the 2127 enrolled patients, 1682 (79.1%) had a CD4 cell count available; of which 52% presented with AHD. Of the Mayan population, 65% had AHD. The overall OI incidence was 21%. Histoplasmosis was the most frequent OI (7.9%), followed by TB (7.1%); 94.4% of these infections occurred in patients with a CD4 < 350 cells/mm3. Mortality at 180 days was significantly higher in those with OIs than without OIs (29.7% vs. 5.9%, p < 0.0001). In one year, this program decreased the OI mortality by 7% and increased the OI treatment by 5.1%. Early OI diagnosis and appropriate therapy reduced OI mortality among newly diagnosed HIV patients in Guatemala. Screening for OIs should be considered in all newly diagnosed HIV patients who have a CD4 cell count < 350 cells/mm3 or those without a CD4 cell count available. To improve results, interventions such as early HIV detection and access to flucytosine and liposomal amphotericin B are required.


Introduction
Opportunistic infections (OIs) remain a major cause of death in HIV-infected patients, especially in low-and middle-income countries (LMICs) [1]. Despite the increasing coverage of antiretroviral therapy (ART), 770,000 people died in 2018 [2]. In Latin America (LATAM), these deaths have decreased by 14% since 2010; however, other regions such as eastern and southern Africa have shown a 44% reduction [2]. National and regional data on specific causes of HIV mortality are crucial to develop strategies and to allocate resources effectively.
In 2017, the World Health Organization (WHO) published the first guidelines for managing advanced HIV disease and rapid initiation of antiretroviral therapy [3]. These guidelines recommended a package of interventions oriented to reduce AIDS-related mortality while advising rapid diagnosis and treatment of major OIs. Although these recommendations are focused on people with advanced HIV disease (AHD), there may be benefits of screening strategies that reach all patients at higher risk, such as those newly diagnosed with HIV. Since 2010, the number of new HIV infections has increased by 7% in LATAM [2]. Differences in the burden of OIs have also been described, especially for histoplasmosis, which is an important OI in the region [4,5]. Therefore, we developed a prospective screening study to assess the burden of AHD, the incidence of four OIs, and the mortality at 180 days in a large cohort of newly diagnosed HIV patients in Guatemala.

Study Design and Participants
Patients were enrolled in the OIs program, which was established in Guatemala in 2017. This program encompasses a network of 13 health care facilities (HCFs) and a central Diagnostic Laboratory Hub (DLH) [6]. This work analyzes all newly diagnosed HIV patients enrolled in this program between January 2017 to December 2018 who were over 13 years of age.

Procedures and Definitions
Demographic data were collected in the HCFs using a standard electronic form. Patients were screened for the following OIs: tuberculosis (TB), nontuberculous mycobacteria (NTM), histoplasmosis, and cryptococcosis. Screening was performed independently of the CD4 cell count. The following samples were requested: whole blood in an isolator tube (Abbott Diagnostics, Illinois, Chicago, USA), serum, urine, and sputum. Additional samples were also received based on clinical criteria. Specimens were referred to the DLH to be processed as previously described [6,7]. Laboratory assays included: (i) Smear microscopy; (ii) Sputum culture; (iii) Isolator blood culture; (iv) in-house PCR for M. tuberculosis and H. capsulatum; (v) Detection of urine antigen of H. capsulatum by IgG monoclonal antibodies (Immuno-Mycologics (IMMY), Norman, Oklahoma, USA); and (vi) the cryptococcal antigen lateral flow assay (CrAg) (LFA, IMMY, Norman, Oklahoma, USA). If serum CrAg was positive, a lumbar puncture was advised. The relative diagnostic performance of these tests has been described by Medina et al. [7]. A full screening was considered when the four OIs were analyzed. OI diagnosis required a positive result of at least one of the following techniques: antigen detection, culture, and/or PCR test.
Disseminated histoplasmosis was considered when an Isolator blood culture and/or urine antigen was positive, and cryptococcal meningitis when we detected a positive CrAg test or isolated C. neoformans in cerebrospinal fluid (CSF). AHD was defined as having a CD4 count < 200 cells/mm 3 . CD4 cell counts were obtained by the different HCFs. An available CD4 cell count was defined as a cell count obtained 90 days before or after the moment of the OI screening. Patients from Guatemala City and other cities were categorized as urban while other areas were defined as rural.
Patients were treated in the HCFs and antiretroviral therapy was started in accordance with the national guidelines [8,9].

Ethics Statement
Because this study analyzed data obtained during the implementation of a routine program that provided diagnostic services to newly diagnosed HIV patients, informed consent was not requested for the OI screening; however, a written informed consent for HIV testing was obtained by the HCFs of the network. For the analysis, no personally identifiable data information was collected, and confidentiality of patients was assured.
As expected, the incidence of an OI was inversely correlated with CD4 cell count (Table 2). Histoplasmosis was the most frequent AIDS-defining illness. The difference between histoplasmosis and TB incidence was statistically significant in patients with CD4 cell counts < 50 cells/mm 3 (19.7% vs. 11.5%, p = 0.003). Among those with AHD, the incidence of OIs was 30.3%. In patients who had a CD4 cell count ≥ 350 cells/mm 3 , TB was more frequent than histoplasmosis (2.9% vs. 1.1%, p = 0.0807) ( Table 2). We analyzed, by means of a ROC curve, OIs (Yes/No) against CD4 cell counts to determine the best threshold to screen patients for OIs. The AUC was 0.771 (95%CI; 0.741-0.801). Table 3 shows the sensitivity and specificity of the different thresholds. Almost 95% of the OIs investigated occurred in patients with <350 CD4/mm 3 . Fifteen OIs were diagnosed in patients with ≥350 CD4 cells/mm3: 10 TB, 4 histoplasmosis, and 1 cryptococcal disease. Eighty-one (63.3%) of 128 patients with disseminated histoplasmosis had a CD4 cell count available. If histoplasmosis screening had been limited to patients with CD4 cell counts < 100 or <200 CD4 cells/mm 3 , 16 (19.7%) and 6 (7.4%) cases would have been missed, respectively. For cryptococcal disease, 5 (13.8%) and 1 (2.8%) cases would have been missed, respectively. A total of 117 (30.4%) OIs were diagnosed in patients without a CD4 cell count result available.

Discussion
This study analyzes the situation of newly diagnosed HIV patients in Guatemala. The data are able to provide robust evidence since they account for 58.3% (2127 out of 3646) of the new HIV infections reported by the national HIV program during 2017-2018. The proportion of the newly diagnosed HIV patients presenting with AHD was 52%, which was 6% higher than the estimated frequency by UNAIDS of 46% [2]. In other LATAM countries, AHD ranged from 20 to 40% [2]. Several studies in Asia (36.3%), Ethiopia (39%), South Africa (35.6%), and Rwanda (29.4%) also showed lower rates of AHD [10][11][12][13][14]. Therefore, Guatemala has one of the highest rates of newly diagnosed HIV patients presenting with AHD in the world. This rate was especially high in the Mayan population with 65.8%. A previous study in Guatemala found lower levels of HIV knowledge in the Mayan population compared with Ladinos [15]. Thus, limited information and structural disadvantages could explain the late diagnosis. We also found that heterosexual patients were more likely to be associated with AHD at the moment of diagnosis compared with MSM, similar to results found in other studies [16,17]. This is possibly due to awareness campaigns and active HIV screening programs implemented in recent years that have promoted an earlier diagnosis in MSM. Overall, the high frequency of AHD underscores the urgent need for national campaigns and adapted HIV testing strategies.
To diagnose OIs in newly diagnosed HIV patients, the WHO recommends screening for cryptococcal antigen in patients with CD4 cell counts < 100 cells/mm 3 and routine clinical evaluation for TB [3,18]; however, we screened all patients irrespective of their CD4 cell count. Our results showed that 27.9% of OIs occurred in patients with CD4 cell counts >100 cells/mm 3 ( Table 2). The ROC analysis using CD4 cell counts <200 cells/mm 3 as the screening threshold showed a sensitivity of 85.1% and in patients with <350 cells/mm 3 a sensitivity of 94.4%. The number needed to test is six patients to diagnose one case of OI at CD4 count of 100-200 cells/mm 3 and 15 at a CD4 count of 200-350 cells/mm 3 . For this reason, we cannot recommend a CD4 cell count <200 cells/mm 3 as the screening threshold since almost 15% of the OIs would have been missed. Considering that OIs are usually life-threatening diseases, we find it reasonable to spend $7.5 on cryptococcal antigen tests and $13 on histoplasmosis testing. Furthermore, TB should be investigated independently of the CD4 cell count.
Histoplasmosis was the most common OI with an overall incidence of 7.9% rising to 19.7% in patients with CD4 cell counts < 50 cells/mm 3 . Guatemala is known to be a hyperendemic area for histoplasmosis [4,5,19]. Here, we found almost double the incidence than the estimated in a previous study of the histoplasmosis burden in LATAM (4.1% vs. 7.9%) [4]. In June 2020, the Pan American Health Organization (PAHO) published guidelines for diagnosing and managing disseminated histoplasmosis in people living with HIV [20]. This document recommends urine antigen detection as the reference technique to diagnose disseminated histoplasmosis [20]. In Guatemala, a comparative study of the laboratory assays showed the highest rate of detection for histoplasmosis with the urine antigen test [7]. In addition, it is clinically difficult to determine whether the patient has histoplasmosis or tuberculosis. Therefore, the screening approach provides the means to differentiate both OIs. Concerning cryptococcal disease, current estimations in LATAM are limited. A previous study of the global burden of HIV-associated cryptococcal meningitis estimated the overall prevalence of cryptococcal antigen in patients with CD4 cell counts <100 cells/mm 3 to be 6% [21]. Here, we found that 8.2% of AHD patients had a positive serum CrAg test. Hence, it seems that the burden of histoplasmosis and cryptococcal disease have been underestimated in the region. For TB cases, a previous report by the Pan American Health Organization (PAHO) estimated an incidence of 9.9% among HIV patients in Guatemala in 2014 [22] This was 1.95% higher than the overall TB incidence found in this study (9.9% vs. 7.1%). In our cohort, the incidence of TB was 0.8% lower than histoplasmosis. Considering that TB is more frequent than histoplasmosis in urban areas and one of the largest HCFs, which is located in Guatemala City, did not participated in this study, we can hypothesize that the incidence of TB might be higher.
Mortality among newly diagnosed HIV patients was high with 52.1% of deaths attributed to the OIs screened. A substantial number of patients also died with an unknown diagnosis. This should be improved with the expansion of the diagnostic portfolio. It is important to highlight that the diagnosis has to be made quickly since 53.9% of deaths occurred in the first 30 days. The highest mortality rate was observed in patients with multiple OIs (48.4%). Contributing factors to this increased mortality could include severe immunosuppression (96% had AHD), complicated management, and drug-drug interactions [23]. Histoplasmosis was also associated with a high mortality, similar to previous analyses (28.3%) [24,25]. We found a 7% reduction in the OI mortality in 2018 compared to 2017, with a simultaneous increase in the OI treatment (5.1%). This finding should be attributed to the early diagnostic capacity provided by the OI program. Nevertheless, mortality of cryptococcal meningitis was 34%. It is well known that amphotericin B plus flucytosine decreases cryptococcal meningitis mortality [26,27], yet flucytosine is not available in Guatemala. Therefore, its introduction into the country should be a priority. In addition, the mortality rate in patients with cryptococcal antigenemia without a lumbar puncture (LP) was similar to those with meningitis (37% vs. 34%), suggesting that better management of these patients should be instituted. Reasons for not realizing LP in these patients was not recorded. Since the introduction of the Histoplasma urine antigen test, the associated mortality has decreased by 8.3% (From 36.6% in a reference HCF to 28.3% in the network) [28]. However, liposomal amphotericin B is not available in Guatemala, and mortality could be further decreased with its introduction. Concerning NTM, 15 cases were diagnosed with a high mortality rate (40%). Here, NTM identification requires the growth of the microorganism, which can take several weeks. Thus, rapid methods such as GenoType CMdirect (Hain Lifescience) that can be performed from direct specimens would be required.
The present study has its limitations. The OI status was based on the available laboratory techniques in this program, but other OIs have been missed. This will have led to an underestimation of the burden of OIs. Other factors such as ARV treatment were not determined. The integration of all HCFs in the network will improve the accuracy of the data. Despite the limitations, our findings show that Guatemala has one of the highest rates of AHD in newly diagnosed HIV patients, the burden of OIs among newly diagnosed HIV patients is substantial, the burden of fungal infections has been previously underestimated, and access to rapid diagnosis of OIs has decreased the overall mortality by 7% in a single year, showing that it is an essential tool to achieve the goal of eliminating AIDS and reducing deaths.

Institutional Review Board Statement:
The study was conducted according to the guidelines of the Declaration of Helsinki; it was not subject to IRB review because it analyzed data obtained of a routine program that provided diagnostic services, and no human experiments were performed.
Informed Consent Statement: Informed consent was not requested for the OI screening, because it was implemented as a routine service; a written informed consent for HIV testing was obtained by the HCFs of the network.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.