We have shown that splenomegaly among patients with primary L. donovani
VL was strongly associated with a much higher relative risk of VL relapse. Primary VL patients presenting with splenomegaly of Hackett Grade ≥3 had almost four-fold higher odds of subsequent relapse than patients with no enlargement of the spleen, and patients discharged with greatly enlarged spleens had greater than five-fold higher odds of relapse. The latter, but not the former, association remained evident after excluding from our analysis all patients who had no palpable spleen on admission (). No other clinical characteristics of primary VL patients emerged as strong risk factors for VL relapse, although infants appeared to be more susceptible as reported for L. chagasi
Splenomegaly on admission may indicate a combination of severity of illness, parasite burden, and severity of immunosuppression. Splenomegaly on discharge suggests the patient has not responded adequately to treatment, and may either harbour a significant parasite burden or may still be immunosuppressed by the disease. Splenomegaly is one of the classical signs of VL, reported globally as present in >90% of VL patients 
. Extremely enlarged spleen on admission is a sign of advanced disease and is a risk factor for death 
. The spleen in L. donovani
infection is infiltrated by parasitized macrophages as well as plasma cells, immune complexes and other components of immune response, leading to hyperplasia of reticulo-endothelial cells and enlargement of the organ 
. A chronic inflammatory state mediated mainly by TNF results in architectural damage and immunological dysfunction 
. Particularly important for treatment outcome is loss of spleen marginal zone macrophages, which play an important role in capturing blood-borne pathogens; during enlargement, the spleen's protective role is reduced 
. Through clinical and pathophysiological observation, reduction of spleen size is recognised as one of the most important signs of successful treatment, and splenectomy has traditionally been practiced in splenomegalic patients who have frequent relapses despite appropriate treatment 
. However, according to the WHO Manual on Visceral Leishmaniasis, a completely unpalpable spleen is not considered necessary to classify a patient as cured, (“persistently enlarged spleen is no cause for concern provided the patient's other symptoms are improving”) 
Severe anaemia is associated with the increased risk of death among VL patients 
and, apart from other reasons (bone marrow suppression, malnutrition and generalised inflammation), is related to intensive destruction of erythrocytes in enlarged spleens 
. Thus, the correlation between marked splenomegaly and anaemia found in our data would be expected. However, other indicators of advanced disease on admission, such as severe malnutrition and general weakness expressed as walking status, were not associated with relapse in our patients. Weight gain is a sign of clinical response to treatment, and mean percentage weight gain was marginally lower in the group of patients who experienced relapse. However, we note that discharge weight data were missing for 50% of patients. The mean percentage gain in haemoglobin level was the same in both groups, although data were missing for 80% of patients. Other signs of clinical response, such as improvement of general status and becoming afebrile, were not recorded in our database.
We found that 17-day SSG/PM combination therapy was associated with two-fold higher odds of relapse than 30-day SSG monotherapy. The joinpoint analysis showed an upward trend in VL relapse admissions as a proportion of primary VL admissions (), which might be interpreted as a consequence of the introduction of SSG/PM as first-line treatment for primary VL between 2001 and 2003. However, this apparent trend was driven by the relatively high proportion of VL relapse patients treated in the last two years (2006–2007), and the risk factor analyses showed no discernible trend. The 17-day SSG/PM regimen was introduced on the basis of evidence from several trials 
, and in a retrospective study (2002–2005) based on MSF treatment data from Southern Sudan, the SSG/PM regimen was found to give better survival and cure rates than SSG alone 
. Preliminary analysis of a phase III clinical trial by the Drugs for Neglected Diseases Initiative (DNDi) indicated as yet no difference in cure rates between 17 days of SSG/PM and 30 days of SSG [DNDi - unpublished data]; final results of the trial are due in 2010 
. It could be that the very patients who were saved from dying by SSG/PM may be those who relapsed. This phenomenon probably occurred during our study of miltefosine vs
SSG in Ethiopia: miltefosine, being a safer drug than SSG, was associated with a far lower death rate, yet a far higher relapse rate 
. However, not all previously-reported risk factors for death were evident as risk factors for relapse (possibly because drug toxicity is a primary cause of death) 
, and we cannot discount a possible role of shorter duration of treatment in increasing risk of relapse 
Our reported association between SSG/PM and risk of VL relapse was likely to be residually confounded, and possibly biased by missing data, because SSG/PM was used at permanent MSF treatment centres, whereas SSG monotherapy was used at temporary (seasonal) outreach sites. Access to care in the event of VL relapse was much easier at the permanent sites, some of which have even benefited from public transport since 2005. Temporary sites were also less able to diagnose relapses, because the splenic or lymph node aspirates which are necessary to make the diagnosis of relapse could only be carried out at the main treatment centres. These factors, together with better record-keeping, probably explain the apparently higher risk of relapse associated with treatment at Lankien.
The use of SSG/PM during the latter half of the period in our study coincided with a series of political agreements (between 2003 and 2005), which brought a tentative ceasefire to Southern Sudan after decades of conflict. Hence, the crude upward trend seen in MSF's summary data is probably a consequence of easier access to treatment, as reflected in the trend towards shorter ‘time-to-presentation’ for VL patients (). In the past, when access to care was restricted, patients with relapse may have died without reaching a treatment centre. HIV infection was unlikely to be a factor in VL relapse during most of the period in our study. An unlinked screening study of 206 VL patients in Lankien in 2002 revealed only 1 HIV-positive case (0.5%); or 1 in 36 adult patients (2.8%) [MSF - unpublished data]. However, the number of VL patients has declined in recent years (from 4,172 in 2003 to 101 in 2008), while large numbers of refugees have begun to return to Southern Sudan from places with higher HIV prevalence (Ethiopia, Kenya, Khartoum). Hence we might expect the proportion of VL relapses attributable to HIV co-infection to increase. Recent (2008) data from Nasir, where MSF began routine HIV testing and anti-retroviral therapy in adult VL patients, showed that 25% (5/20) were HIV co-infected [MSF - unpublished data].
Our study was the largest retrospective analysis of VL relapse globally. The difficult conditions under which the data were collected gave rise to some limitations, principally missing treatment records, missing data and, for this study, our inability to link all VL relapse patients with their record of treatment for primary VL. This was mainly because the majority of patients did not present the identification card given to them when they were discharged from treatment for primary VL. It is difficult to assess the impact of missing records and missing data on our outcomes, although we have no reason to suspect these were a source of significant bias in our analysis of clinical risk factors. Some of the primary VL patients whom we classified as “primary VL patients who did not subsequently relapse” may have had an untreated or unrecorded relapse, and some patients who had been previously treated may have been diagnosed as primary VL if the patient did not recall their primary episode. These two misclassifications would lead to under-estimation of risk factors for relapse. The 5 patients (3%) re-admitted soon after discharge (19 to 31 days) may have been treatment failures, rather than relapses. Through our sensitivity analysis we attempted to adjust for those patients with no palpable spleen, who were possibly falsely diagnosed as VL patients (hence at zero risk of relapse). However, in a setting with high prevalence of other endemic diseases which cause splenomegaly (malaria, typhoid, schistosomiasis, brucellosis, and liver cirrhosis), some patients with splenomegaly may also not have had primary VL. We could not conduct a further sensitivity analysis, based on the actual diagnostic method for each patient (parasitological, immunological, clinical), because this could not be deduced from the available data. We noted that relapse VL patients for whom we could not identify a previous treatment record had less severe splenomegaly on re-admission than relapse VL patients whose previous treatment data were used in our analysis. However, spleen size on admission for relapse VL was not correlated with spleen size at end of treatment for primary VL, hence this difference would not have led to an over-estimate of the association between splenomegaly at end of treatment for primary VL and subsequent relapse.
Our finding that SSG/PM is associated with increased risk of relapse is likely to be confounded by improvements in access to treatment which coincided with the introduction of this shorter combination drug regimen, and may reflect better survival rates compared with SSG monotherapy. We await a definitive answer from a randomised controlled trial of SSG/PM vs SSG currently underway in East Africa. Meanwhile, our finding that splenomegaly is associated with increased risk of VL relapse could contribute to revised guidelines for clinical assessment of VL patients prior to discharge.