Expression profiling of Sudanese visceral leishmaniasis patients pre- and post-treatment with sodium stibogluconate

Visceral leishmaniasis (VL) in Sudan caused by Leishmania donovani is fatal in susceptible individuals if untreated. Treatment with Sodium Stibogluconate (SSG) leads to post kala azar dermal leishmaniasis (PKDL) in 58% of patients. Here Affymetrix microarrays were used to identify genes differentially expressed in lymph nodes (N=9 paired samples) pre- and post-treatment with SSG. Using the Bioconductor package limma, 438 genes from 28,869 post quality-control probe-sets were differentially expressed (Pnominal≤0.02) post- versus pre-treatment. Canonical pathway analysis using Ingenuity Pathway Analysis identified “Role of NFAT in Regulation of Immune Response” (Pnominal=1.35x10-5; PBH-adjusted=4.79x10-3), “B Cell Development” (Pnominal=2.04x10-4; PBH-adjusted=0.024), “Fcγ Receptor-mediated Phagocytosis in Macrophages and Monocytes” (Pnominal=2.04x10-4; PBH-adjusted=0.024), and “OX40 Signaling” (Pnominal=2.82x10-4; PBH-adjusted=0.025) as pathways differentially regulated post- versus pre-treatment. Major network hub genes included TP53, FN1, MYC, BCL2, JUN, SYK, RUNX2, MMP1 and ACTA2. Top endogenous upstream regulators included IL-7 (P=2.28x10-6), TNF (P=4.26x10-6), APP (P=4.23x10-5) and SPI1/PI.1 (P=1.17x10-7). Top predicted chemical drug regulators included the flavonoid genistein (P=4.56x10-7) and the quinoline alkaloid camptothecin (P=5.14x10-5). These results contribute to our understanding of immuno-pathology associated with VL and response to SSG treatment. Further replication could identify novel therapeutic strategies that improve on SSG treatment and reduce the likelihood of progression to PKDL. Samples were collected in Kassab Hospital, El-Gadarif State, Sudan. Active VL cases were diagnosed by experienced clinicians on the basis of clinical signs, including fever, hepatosplenomegaly, anaemia, leukopenia, pancytopenia, and by microscopic demonstration of Leishmania amastigotes in slide films prepared from inguinal lymph node aspirates. VL patients received the standard daily dose of 20 mg SSG per kg body weight for 30 days. Cure was confirmed by demonstration that post-treatment lymph node aspirates were negative for parasites by microscopy. RNA was extracted from inguinal lymph node aspirates taken from 9 paired VL patients (N=7 males, aged 7 to 19 years; N=2 females, both aged 8 years) before and after treatment (i.e. 18 samples). Aspirate volume was equivalent to the volume of an 18Gx2.5cm fine needle. Aspirates were directly injected into 700uL of RLT buffer (RNeasy Micro Kit; QIAGEN, UK), and RNA prepared using these kits in accordance with the manufacturer´s instruction. RNA integrity and purity were checked using a 2100 Bioanalyzer with the RNA 6000 Nano and Pico LabChip kit (Agilent Technologies, Massy, France). All samples had RNA integrity (RIN) values >7.