Incidence of WHO Stage 3 and 4 Conditions following Initiation of Anti-Retroviral Therapy in Resource Limited Settings

Objectives To determine the incidence of WHO clinical stage 3 and 4 conditions during early anti-retroviral therapy (ART) in resource limited settings (RLS). Design/Setting A descriptive analysis of routine program data collected prospectively from 25 Médecins Sans Frontières supported HIV treatment programs in eight countries between 2002 and 2010. Subjects/Participants 35,349 study participants with median follow-up on ART of 1.33 years (IQR 0.51–2.41). Outcome Measures Incidence in 100 person-years of WHO stage 3 or 4 conditions during 5 periods after ART initiation. Diagnoses of conditions were made according to WHO criteria and relied upon clinical assessments supported by basic laboratory investigations. Results The incidence of any WHO clinical stage 3 or 4 condition over 3 years was 40.02 per 100 person-years (31.77 for stage 3 and 8.25 for stage 4). The incidence of stage 3 and 4 conditions fell by over 97% between months 0–3 and months 25–36 (77.81 to 2.40 for stage 3 and 28.70 to 0.64 for stage 4). During months 0–3 pulmonary tuberculosis was the most common condition diagnosed in adults (incidence 22.24 per 100 person-years) and children aged 5–14 years (25.76) and oral candidiasis was the most common in children <5 years (25.79). Overall incidences were higher in Africa compared with Asia (43.98 versus 12.97 for stage 3 and 8.98 versus 7.05 for stage 4 conditions, p<0.001). Pulmonary tuberculosis, weight loss, oral and oesophageal candidiasis, chronic diarrhoea, HIV wasting syndrome and severe bacterial infections were more common in Africa. Extra-pulmonary tuberculosis, non-tuberculous mycobacterial infection, cryptococcosis, penicilliosis and toxoplasmosis were more common in Asia. Conclusions The incidence of WHO stage 3 and 4 conditions during the early period after ART initiation in RLS is high, but greatly reduces over time. This is likely due to both the benefits of ART and deaths of the sickest patients occurring shortly after ART initiation. Access to appropriate disease prevention tools prior to ART, and early initiation of ART, are important for their prevention.


Introduction
Efforts to scale up provision of anti-retroviral therapy (ART) in resource-limited settings (RLS) have dramatically increased over the past decade [1,2]. A vital role of such programs is diagnosis, treatment and prevention of HIV associated conditions which contribute significantly to morbidity and mortality in patients on ART in RLS [2,3,4,5].
In developed countries ART reduces the incidence of HIV associated conditions [6,7,8,9]. In RLS there is a lack of data regarding the frequency and relative importance of common HIV associated conditions after commencement of ART. It is also unclear whether there are differences in disease incidence rates between regions and age groups.
A more detailed understanding of the incidence of specific HIV associated conditions after ART initiation will aid clinicians in management of patients during ART and assist program planners in strategic decision making and resource allocation for prevention, diagnosis and treatment of these conditions. Therefore, this study aims to describe the incidence of WHO Stage 3 and 4 conditions for three years following ART initiation in patients attending Médecins Sans Frontières (MSF) supported treatment programs in RLS and to examine how this is influenced by region and age.

Ethics Statement
The Monash University Human Research Ethics Committee, Alfred Health Human Ethics Committee, and MSF Human Ethics committee approved the study.

Study Setting and Population
We analysed routine clinical data collected prospectively between 2002 and 2010 from 25 MSF supported HIV treatment programs in ten countries (Democratic Republic of Congo, Ethiopia, India, Ivory Coast, Moldova, Myanmar, Nigeria, Republic of Congo, Zambia, and Zimbabwe).
Adults and children who commenced ART without previously taking antiretrovirals and could contribute at least 3 months of follow-up data were eligible for inclusion. Patients were stratified into the following age groups: ,5 years, 5-14 years, $15 years. Children ,2 years of age with unknown HIV status who were receiving ART were excluded.
HIV associated clinical conditions diagnosis and treatment protocols, adherence counselling, patient follow-up, data collection and monitoring, laboratory protocols, and drug procurement and supply mechanisms were standardised across MSF programmes [10]. Treatment sites included hospital settings and peripheral health clinics.
Diagnoses of HIV-associated clinical conditions were usually made according to WHO criteria for individual conditions [11] by treating clinicians (medical doctors and clinical officers). Diagnoses relied on clinical assessments supported by basic laboratory investigations, including biochemistry, haematology, sputum microscopy for acid fast bacilli (AFB) stains, India ink examination of cerebro-spinal fluid and chest x-ray. More sophisticated investigations such as cryptococcal antigen assay and abdominal ultrasound were available at limited sites.
ART regimens were usually non-nucleoside reverse-transcriptase inhibitor (NNRTI) based. Eligibility criteria for ART and first-line regimens were standardized and based on WHO guidelines [11,12]. Clinical consultations and intensive adherence counselling sessions were provided before and during ART. Daily co-trimoxazole prophylaxis was given to all patients with clinical stage 2, 3 and 4 disease or those with CD4 cell count ,350 cells/ mm 3 . Patients did not routinely receive prophylaxis for tuberculosis (TB) or Mycobacterium Avium Complex (MAC), apart from those in Myanmar programmes where MAC prophylaxis was generally provided for patients with CD4,50 cells/mm 3 . CD4 counts were usually monitored using automated methods but HIV viral load was not systematically monitored. All treatment was free.

Data Collection
Information about medical history and sociodemographic characteristics were collected at the initial consultation. Clinical and therapeutic information, including any new HIV associated clinical conditions, weight, and duration of follow up was recorded at subsequent consultations. All information was collected on standard forms and entered into FUCHIA software on site (Follow-up and Care of HIV Infection and AIDS, Epicentre).

Analysis
Categorical variables were summarised using frequency and percentage and analysed using a chi-square test. Continuous variables were first tested for normality. As all were significantly skewed they were summarised using median and inter-quartile range (IQR) and analysed using a Wilcoxon rank-sum test.
Incidence rates were designed a priori to be reported as crude, unadjusted rates. Incidence rates for any WHO stage 3 or 4 condition were calculated for five periods after ART initiation (0-3, 4-6, 7-12, 13-24 and 25-36 months) by dividing the number of occurrences of any stage 3 or 4 condition by the number of personyears at risk, within that particular time period. Patients were censored at death, loss to follow-up, or, where no disease event was recorded within a particular time period, at 3, 6, 12, 24 or 36 months respectively after ART initiation. Patients missing an appointment by $2 months were considered lost to follow-up and censored on the day of their last visit. All incidence estimates are conditional on patients being alive and on treatment at the beginning of the period, and as they are crude and unadjusted, and the groups are not independent, direct comparisons between time-periods should be interpreted with caution. As we did not evaluate whether incidence rates might have declined due to deaths in the prior time period, we cannot state the degree to which changing rates are due to the impact of ART or to the death of severely ill persons in the prior time period.
Disease specific incidence rates were calculated for the same time periods by dividing the number of first diagnoses of each WHO stage 3 or 4 condition by the number of person-years at risk. These analyses considered only the WHO stage 3 or 4 condition of interest and data was censored as described above for any WHO stage 3 or 4 incidence.
Analyses were also performed by region (Africa and Asia) and by age group. Data from Moldova (Eastern Europe) was excluded from the regional analysis due to the small number of patients. Confidence intervals were calculated for all rates.
Data were extracted from individual program FUCHIA databases, de-identified and analysed using Stata version 12 (StataCorp, College Station, Texas, USA).

Patients
A cohort of 36,664 patients initiated ART treatment. Table 1 shows the baseline characteristics of 34,749 patients who contributed at least 3 months follow-up and were included in the analysis. Sixty-two percent of these patients were from African programmes and 36% were from Asian programmes. Eighty-nine percent of patients were aged $15 years, 5% were aged 5-14 years, and 6% were aged ,5 years; there were proportionally less adults and more children in African compared to Asian programmes (p,0.001). The median time on ART was 1.33 years, with patients attending Asian programs followed longer than those attending African programmes (median 1.6 years and 1.1 years, respectively, p,0.001). A larger proportion of patients attending African sites was female (67% compared to 44%, p,0.001). The median CD4 level at ART commencement was higher in African patients than in Asian patients for all age-groups (p,0.001).
A further 600 patients with 3 months follow-up from the Moldova programme in Eastern Europe were included in all analyses except the regional analysis. Ninety-three percent of Moldovan patients were aged $15 years, 0.1% were aged 5-14 years and 5.8% were aged ,5 years. The median time on ART was 0.58 years, 67.8% of patients were male and the median CD4 level at ART commencement for adults was 385 (IQR: 201-575) cells/mm 3 . We did not detect significant differences in the incidence of any stage 4 condition between patients aged 5-14 years and patients $15 years (p = 0.25).

Incidence of any WHO Stage 3 or 4 Conditions
Regional differences were apparent in the overall disease burden due to both stage 3 and stage 4 conditions. The incidence of any WHO stage 3 condition was nearly 4 times higher in Africa than in Asia, with 43.98 (95% CI 43.29, 44.68) and 12.97 (95% CI 12.52, 13.42) episodes per 100 person-years respectively (p,0.001). For any WHO stage 4 condition, the overall incidence was 8.98 (95% CI 8.67, 9.30) and 7.05 (95% CI 6.73, 7.39) diagnoses per 100 person-years in Africa and Asia, respectively (p,0.001). Tables 2 and 3 show the incidence over 3 years follow-up for new WHO stage 3 and stage 4 conditions. The maximum incidence for each stage 3 and stage 4 condition occurred during months 0-3 and decreased over time. Of the WHO stage 3 conditions, oral candidiasis and pulmonary TB had the highest initial incidence at 22.07 (95% CI 20.92, 23.28) and 21.96 (95% CI 20.81, 23.17) episodes per 100 person-years, respectively. The incidence of these conditions fell by 91% and 87%, respectively, by 3 years of follow-up. For the remaining stage 3 conditions shown, the reduction in disease specific incidence during follow-up ranged from 82% for other severe bacterial infections and weight loss .10% of body weight to 93% for unexplained anaemia, neutropenia or thrombocytopenia. Of the WHO stage 4 conditions, extra-pulmonary TB and candidiasis of the oesophagus, trachea, bronchi or lungs had the highest initial incidence, at 5.30 (95% CI 4.75, 5.90) and 4.67 (95% CI 4.16, 5.25) episodes per 100 person-years during months 0-3, respectively ( Table 3). The incidence of these conditions fell by 82% and 92%, respectively, by 3 years of follow-up. For the remaining WHO stage 4 conditions shown, the reduction in disease specific incidence during follow-up ranged from 82% for mucocutaneous herpes simplex infection to 97% for nontuberculous mycobacterial infection and Pneumocystis jiroveci pneumonia.

Regional Differences in Disease Specific Incidence
The disease specific incidence of individual WHO stage 3 conditions in months 0-3 was higher in Africa than in Asia for all conditions except oral hairy leucoplakia (Table 4). In Africa, pulmonary TB had the highest incidence of all WHO stage 3 conditions and this was 3 times greater than the incidence in Asia, with 30.45 (95% CI 28.68, 32.33) compared to 9.90 (95% CI 8.73, 11.23) episodes per 100 person-years in months 0-3. The incidence of oral candidiasis and weight loss .10% of body weight were also high in Africa with 29.84 (95% CI 28.09, 31.70) and 11.78 (95% CI 10.71, 12.96) episodes per 100 person-years in months 0-3, respectively. In Asia, oral candidiasis had the highest incidence of all WHO stage 3 conditions, with 10.32 (95% CI 9.12, 11.67) episodes per 100 person-years in months 0-3. The incidence of oral hairy leucoplakia was also high in Asia, with 9.41 (95% CI 8.27, 10.71) episodes per 100 person-years in months 0-3. The disease specific incidence of WHO stage 4 conditions in months 0-3 was higher in Asia for 60% of conditions (Table 5). These included extra-pulmonary TB, non tuberculous mycobacterial infections, toxoplasmosis, pneumocystis jiroveci pneumonia and cryptococcosis. Penicilliosis occurred exclusively in Asia. HIV wasting syndrome, candidiasis of the oesophagus, trachea, bronchi or lungs, chronic mucocutaneous herpes simplex virus infection, Kaposi's sarcoma, encephalopathy and lymphoma were the WHO stage 4 conditions with higher incidence in Africa than Asia.
These regional differences in disease specific incidence continued over time, but by 24-36 months follow-up post ART initiation, the only differences between regions were for the WHO stage 3 conditions pulmonary TB, oral candidiasis, weight loss and chronic diarrhoea which remained higher in Africa than Asia.

Discussion
We demonstrated a high overall incidence of any WHO stage 3 or 4 condition (40.02 episodes per 100 person-years) in the 3 years following ART initiation in patients in RLS. In the 3 months immediately following ART initiation, the incidence of any WHO stage 3 or 4 condition was nearly 14 times higher than that reported for a resource-rich setting by the Swiss HIV Cohort Study (106.5 episodes compared with 7.7 episodes per 100 personyears) [6]. However, we also showed that there were significant reductions, particularly in the first 12 months, in the incidence of new WHO stage 3 or 4 conditions during the first 36 months of ART. Both stage 3 and 4 conditions reduced in overall incidence by more than 90% in the first year and reductions in disease specific incidence rates of the conditions with highest initial incidence ranged from 82% to 97%. These striking decreases in     incidence during the first year of ART are similar to those observed in patients on ART in high income countries [6,9,13]. As our analyses are not adjusted for death and loss to follow-up in the cohort, the absolute reductions in incidence rates cannot be ascribed to the beneficial effects of ART alone [14]. Nevertheless it is known that the immune restorative effects of ART reduce the incidence of new or recurrent WHO stage 3 and 4 conditions [6,7,8,9] and it is likely that ART has played a significant role in their reductions in our cohort. This emphasizes the importance of access to early ART initiation for those eligible to reduce the impact of HIV related conditions during early ART in RLS. This requires increased efforts to enable early diagnosis and accurate clinical and immunological staging of HIV infected patients, and their rapid access to effective, non-toxic, free and sustained ART in these settings. The considerable incidence of HIV associated clinical conditions during early ART also emphasizes the importance of increased disease prevention efforts prior to ART commencement. Such interventions include isoniazid chemophrophylaxis for TB [15,16], fluconazole prophylaxis for those who are cryptococcal antigen negative and have delays in ART initiation [17,18] and cotrimoxazole therapy for PCP, toxoplasmosis, bacterial sepsis and malaria [19,20]. Resources and improved tools are also required to facilitate early and improved diagnosis of opportunistic infections, as well as effective, adapted and affordable treatments to minimize their impact on morbidity and mortality.
We defined the burden of different WHO stage 3 and 4 conditions stratified by age and region. This provides information for clinicians to use in determining which conditions to focus on for prevention, early diagnosis and treatment. It is also vital information for programme planners to guide allocation of resources required for disease management in patients on ART, and for strategic planning and resource prioritization. For example, age-specific and regional incidence rates of TB in patients on ART can aid strategic decision making about which patient populations to prioritise in implementing isoniazid preventive therapy (IPT) if universal IPT is not chosen or possible. Also, knowing specific disease incidences can aid in decision making about programme drug formularies.
TB and candidiasis were the most common conditions seen in our RLS cohort. TB is a leading cause of disease in HIV infected patients and is the leading cause of HIV-related deaths [2,17,21]. Our results re-affirm the importance of TB as a pathogen in HIV infected patients treated with ART. Importantly, our results support other studies in suggesting that ART alone reduces the incidence of TB [22,23,24]. Provision of IPT can further reduce the impact of TB whilst on ART [15,25] and is currently recommended [16]. Likewise our study showed ART is associated with a reduction in the incidence of candidiasis. Although a recent study from Uganda showed that giving fluconazole prophylaxis to those with a CD4 count ,200 cells/mm 3 can further reduce the impact of candidiasis both pre and post ART [17], it did not reduce overall mortality, and is currently not recommended by WHO [18]. Important contributions to the disease burden in our study were also made by potentially preventable stage 4 conditions such as non-tuberculous mycobacteria, cryptococcosis, pneumocystis and toxoplasmosis. The most common conditions differed from those in a resource rich setting where the oseophageal candidiasis, PCP and cytomegalovirus disease were the most frequently reported conditions during the first 6 months of ART [6]. There were important regional differences in the overall and disease specific incidences of any stage 3 and 4 condition following ART initiation. The incidence of any stage 3 HIV associated condition was almost 4 times higher in Africa than Asia despite African patients being less immunosuppressed overall at baseline. This may be due to increased exposure to the relevant pathogens in Africa or less likely, to improved diagnosis in Africa [22,26]. Some individual stage 4 conditions were more common in Asian programmes, particularly in the first 3 months after ART initiation, likely due to the higher rate of immunosuppression of these patients at baseline, and differential prevalence of specific pathogens. For example, penicilliosis is not endemic in Africa.
The incidence of any first stage 3 condition did not differ by age however there were important differences in the incidence of individual conditions by age. Pulmonary TB was diagnosed less commonly in children ,5 years, although this may be underestimated given the difficulty of diagnosis in this age-group. Additionally, severe bacterial pneumonia was more common in children ,15 years compared with adults.
There were significant age-related differences in the overall incidences of stage 4 conditions that may be explained by the relative level of baseline immunodeficiency. HIV-infected children ,5 years usually have rapidly progressive disease with high mortality [27]. This increased immunodeficiency may explain the higher incidence of stage 4 conditions in this age-group. Conversely, many children in the 5-14 age-group will have been infected peripartum, but survived due to less rapidly progressive disease. This may explain this group's low incidence of stage 4 conditions. In addition, there were more differences seen in the incidence of specific stage 4 conditions between children ,5 years and adults. Children ,5 years were more likely to have HIV wasting compared with adults but less likely to have all other conditions except pneumocystis pneumonia. There were no cases of toxoplasmosis, penicilliosis or Kaposi's sarcoma in patients ,5 years of age. As reported previously [28,29], crytptococcosis was rare under 15 years of age (1 case), supporting recent WHO guidelines recommending against screening and pre-emptive treatment in children due to low prevalence [18].
Information about age-specific incidence rates of various conditions during ART may have important practical applications. For example, TB incidence reduced by 48% during the first 12 months of ART in children ,5 years which was less than the 79% reduction in adults and 77% reduction in children [5][6][7][8][9][10][11][12][13][14] years. This suggests IPT may have relatively greater additional impact in children ,5 years than older age-groups during early ART. In addition, the incidence of bacterial pneumonia during the first 12 months of ART was up to 7 times higher in children than adults, supporting particular attention being paid to the prevention, diagnosis and treatment of this condition in children.
This study has a number of limitations. Firstly, reliance on basic investigations to support clinical assessment in the diagnosis of HIV associated conditions may have resulted in diagnostic inaccuracies. For example TB in children ,5 years old is difficult to confirm with AFB stains alone, and reliance on clinical assessment may have under or overestimated its true incidence. In addition, clinician practices and availability of investigations may have varied between programmes, influencing the relative frequencies of diagnoses. Nevertheless, the results of this study remain relevant because the diagnoses reflect the reality of clinical practice in most RLS where ART is delivered and involves a large cohort of patients. Secondly, the lack of pre-ART data precluded the comparison of incidence rates before and after ART. Thirdly, the Asian data was predominantly from Myanmar programmes so results may not be generalisable across all Asian programmes. Fourthly, we cannot judge the extent to which declining incidence rates are due to ART and care effects; some of these declines may be due to deaths of severely ill persons in the prior time periods. Finally, we were unable to ascertain whether patients lost to follow-up developed any HIV related conditions. Furthermore, as inclusion criteria required at least 3 months of follow-up on ART, patients developing stage 3 and 4 conditions in the first 3 months, which may have increased their risk of lost-to follow-up, could have led to an underestimation of incidence rates in this initial period.

Conclusion
In RLS the incidence of WHO stage 3 and 4 conditions in the early period following ART initiation is high when compared to similar studies in resource-rich settings. However, with early deaths of the sickest patients and with the benefits of ART for the remaining patients, the incidence of HIV associated conditions greatly reduces with time on ART, particularly within the first 12 months. TB and candidiasis account for the greatest disease burden, but many other potentially preventable diseases are also important. Efforts directed towards prevention, early diagnosis and effective treatment of these conditions before and during ART, and the early and accurate assessment for, and initiation of, ART in eligible patients, are important to reduce morbidity during ART in RLS.