[show abstract][hide abstract] ABSTRACT: Healing/protective responses in human visceral leishmaniasis (VL) are associated with stimulation/production of Th1 cytokines, such as interferon IFN-gamma, and conversion in the leishmanin skin test (LST). Such responses were studied for 90 days in 44 adult healthy volunteers from VL non-endemic areas, with no past history of VL/cutaneous leishmaniasis (CL) and LST non-reactivity following injection with one of four doses of Alum-precipitated autoclaved Leishmania major (Alum/ALM) +/- bacille Calmette-Guerin (BCG), a VL candidate vaccine. The vaccine was well tolerated with minimal localized side-effects and without an increase in antileishmanial antibodies or interleukin (IL)-5. Five volunteers (5/44; 11.4%) had significant IFN-gamma production by peripheral blood mononuclear cells (PBMCs) in response to Leishmania antigens in their prevaccination samples (P = 0.001) but were LST non-reactive. On day 45, more than half the volunteers (26/44; 59.0%) had significantly high LST indurations (mean 9.2 +/- 2.7 mm) and high IFN-gamma levels (mean 1008 +/- 395; median 1247 pg/ml). Five volunteers had significant L. donovani antigen-induced IFN-gamma production (mean 873 +/- 290; median 902; P = 0.001), but were non-reactive in LST. An additional five volunteers (5/44; 11.4%) had low IFN-gamma levels (mean 110 +/- 124 pg/ml; median 80) and were non-reactive in LST (induration = 00 mm). The remaining eight volunteers had low IFN-gamma levels, but significant LST induration (mean 10 +/- 2.9 mm; median 11). By day 90 the majority of volunteers (27/44; 61.4%) had significant LST induration (mean 10.8 +/- 9.9 mm; P < 0.001), but low levels of L. donovani antigen-induced IFN-gamma (mean 66.0 +/- 62 pg/ml; P > 0.05). Eleven volunteers (11/44; 25%) had significantly high levels of IFN-gamma and LST induration, while five volunteers had low levels of IFN-gamma (<100 pg/ml) and no LST reactivity (00 mm). One volunteer was lost to follow-up. In conclusion, it is hypothesized that cellular immune responses to human VL are dichotomatous, and that IFN-gamma production and the LST response are not in a causal relationship. Following vaccination and probably cure of VL infection, the IFN-gamma response declines with time while the LST response persists. LST is a simple test that can be used to assess candidate vaccine efficacy.
[show abstract][hide abstract] ABSTRACT: In a longitudinal study in the epidemiology of Leishmania donovani infection in an endemic focus in eastern Sudan, we observed that previous exposure or infection with Leishmania major appeared to protect against visceral leishmaniasis caused by L. donovani. We therefore conducted a study to test the safety and immunogenicity of a vaccine consisting of autoclaved L. major (ALM) plus BCG in inducing protection in vaccinated individuals. Leishmanin-negative healthy Sudanese volunteers were enrolled in the study and were divided into three groups: group (A) received ALM+BCG, group (B) received BCG alone, and group (C) received the vaccine diluent. The subjects were examined for their clinical and immunological responses before intervention, following intervention and 6-8 weeks after vaccination. Vaccinated subjects (group A) developed localized reactions at the sites of vaccine inoculation that ulcerated and healed within 4-6 weeks; 61.6% of them converted to leishmanin reactive following vaccination. Only one subject in group (C) became leishmanin-positive. A total 76.9% of the vaccinated volunteers in group (A) produced significant levels of interferon-gamma in response to L. major antigen. The vaccine produced significant cellular immune responses that may protect against natural challenge. None of the groups had systemic reactions and all the reactions observed in the vaccinated group were comparable with the BCG-vaccinated group.
[show abstract][hide abstract] ABSTRACT: Persistent immunity against Leishmania: infections in humans is mediated predominantly by CD4(+) T cells of the Th1 phenotype. Herein we report the expression cloning of eight Leishmania: Ags using parasite-specific T cell lines derived from an immune donor. The Ags identified by this technique include the flagellar proteins alpha- and beta-tubulin, histone H2b, ribosomal protein S4, malate dehydrogenase, and elongation factor 2, as well as two novel parasite proteins. None of these proteins have been previously reported as T cell-stimulating Ags from Leishmania: beta-tubulin-specific T cell clones generated against Leishmania: major amastigotes responded to Leishmania:-infected macrophages and dendritic cells. IFN-gamma enzyme-linked immunospot analysis demonstrated the presence of T cells specific for several of these Ags in PBMC from self-healing cutaneous leishmaniasis patients infected with either Leishmania: tropica or L. major. The responses elicited by Leishmania: histone H2b were particularly striking in terms of frequency of histone-specific T cells in PBMC (1 T cell of 6000 PBMC) as well as the percentage of responding donors (86%, 6 of 7). Ags identified by T cells from immune donors might constitute potential vaccine candidates for leishmaniasis.
The Journal of Immunology 02/2001; 166(1):498-505. · 5.52 Impact Factor
[show abstract][hide abstract] ABSTRACT: Visceral leishmaniasis is a major cause of morbidity and mortality in the Sudan. Drug treatment is expensive, and drug resistance is becoming increasingly common. Safe, effective, and cheap vaccines are needed. We report the results of a vaccine trial against human visceral leishmaniasis.
We undertook a double-blind randomised trial to test the safety and efficacy of an autoclaved Leishmania major (ALM) promastigote vaccine (1 mg per dose). Of 5093 volunteers screened, 2306 had negative leishmanin skin tests and reciprocal titres of less than 6400 in the direct agglutination test. They were randomly assigned two doses of ALM mixed with BCG or BCG alone. Volunteers were followed up for 2 years. The primary endpoint was clinical visceral leishmaniasis or post-kala-azar dermal leishmaniasis. Analyses were by intention to treat.
Side-effects were confined to the injection site. The cumulative frequency of visceral leishmaniasis at 2 years did not differ significantly between the group assigned ALM plus BCG and that assigned BCG alone (133/1155 [11.5%] vs 141/1151 [12.3%], p=0.6). The vaccine efficacy was 6% (95% CI -18 to 25). The proportion of individuals showing leishmanin skin conversion was significantly higher in the ALM plus BCG group than in the BCG alone group throughout follow-up (303 [30%] vs 72 [7%] at 42 days). Individuals whose leishmanin test converted after vaccination (induration > or =5 mm) had a significantly lower frequency of visceral leishmaniasis than non-responders (27/375 [7.2%] vs 210/1660 [12.7%], p=0.003).
We found no evidence that two doses of ALM plus BCG offered significant protective immunity against visceral leishmaniasis compared with BCG alone. Leishmanin skin conversion with an induration of 5 mm or more in either group was associated with protection from the disease.
The Lancet 11/2000; 356(9241):1565-9. · 39.06 Impact Factor
[show abstract][hide abstract] ABSTRACT: To test the safety and immunogenicity of two doses of autoclaved L. major (ALM) vaccine mixed with BCG.
Kala-azar endemic area of eastern Sudan.
This was a randomised, double blind and BCG controlled phase I/II study.
Eighty healthy volunteers (forty children and forty adults) with no past history of kala-azar, no reactivity to leishmanin antigen and with a reciprocal direct agglutination test (DAT) titre of <200 were recruited. Informed consents were obtained from volunteers or their guardians in case of children.
Conversion in the leishmanin skin and the DAT tests.
Two intra-dermal injections of either ALM+BCG or BCG alone. The injections were three weeks apart.
Side effects were minimal and confined to the injection site, with no significant difference between the ALM+BCG and the BCG alone groups. The leishmanin skin conversion was significantly higher in the ALM+BCG group compared to the BCG alone group (p<0.0005). Furthermore, the Leishmanin skin test conversion was significantly higher in children than adults (p<0.0005). One adult volunteer in the ALM+BCG group converted in both the Leishmanin skin and the DAT tests.
We conclude that two doses of ALM+BCG are safe and immunogenic, especially in children.
East African medical journal 09/2000; 77(9):468-70.
[show abstract][hide abstract] ABSTRACT: In 1994-1996, we studied a group of 58 game wardens stationed in an area known to be highly endemic for visceral leishmaniasis (kala-azar) for evidence of infection with Leishmania donovani. Leishmania DNA was detected by the polymerase chain reaction in the peripheral blood of cases of active kala-azar, former patients with visceral leishmaniasis, patients, and asymptomatic subjects. Using the cloned antigen rk39, antibodies were detected in 44.2% of the game wardens while leishmanin skin test result was positive in 77% of our sample. It was shown that certain tribes from northern Sudan were more likely to develop subclinical infections, while those of the Baria tribe from southern Sudan and those of the Nuba tribe from western Sudan were more likely to develop visceral leishmaniasis. Whether this is due to genetic factors or previous exposure to Leishmania parasites remains to be elucidated.
The American journal of tropical medicine and hygiene 01/2000; 61(6):941-4. · 2.53 Impact Factor
[show abstract][hide abstract] ABSTRACT: The rK39 enzyme-linked immunosorbent assay (ELISA) was compared with the direct agglutination test (DAT) for Leishmania donovani infection in the Sudan. rK39 ELISA proved more sensitive than DAT in diagnosis of kala-azar (93 and 80%, respectively); both tests may remain positive up to 24 months after treatment. For patients with post-kala-azar dermal leishmaniasis and individuals with subclinical infection, rK39 ELISA performed as well as DAT but could detect infection 6 months earlier in approximately 40% of patients. Conversion in DAT and rK39 ELISA also occurred in leishmanin skin test (LST)-positive individuals, suggesting active parasite replication (rK39 is an amastigote antigen) in these presumably immune individuals. In contrast to DAT, rK39 ELISA also detected infection in randomly selected LST-positive individuals (in four of six) and endemicity (LST-negative) controls (in one of five). rK39 ELISA appears more sensitive than DAT and may prove an important tool in epidemiological studies.
[show abstract][hide abstract] ABSTRACT: Almost all (98%) of 1593 visceral leishmaniasis (VL) patients treated with sodium stibogluconate (Pentostam; Wellcome) in Sudan between 1989 and 1995 and follow-up responded well to treatment. However, the other 33 patients, all of whom were seronegative for HIV, showed partial or no response. The two main causes of unresponsiveness were primary drug resistance (39.3%) and low drug dosages given at peripheral dispensaries (30.3%). All of those who had been sub-optimal doses were cured when adequate doses of the drug were given. A third cause was concurrent disease, particularly pulmonary tuberculosis (18%). With treatment of the concurrent disease, patients responded well to Pentostam. Eight patients who failed to respond to repeated courses of Pentostam did not benefit from pentamidine or sterol inhibitors. Three of these patients responded to liposomal amphotericin B, two responded to splenectomy in association with Pentostam therapy, and three died. Pentostam, given in adequate doses, still appears to be the drug of choice for the treatment of VL in the Sudan Liposomal amphotericin B is a suitable second-line drug.
Annals of Tropical Medicine and Parasitology 04/1998; 92(2):151-8. · 1.31 Impact Factor
[show abstract][hide abstract] ABSTRACT: Twelve Leishmania isolates from visceral leishmaniasis patients in eastern Sudan were characterized using isoenzyme analysis, Southern blotting and polymerase chain reaction (PCR) 'fingerprinting'. Isoenzyme analysis revealed the presence of 3 zymodemes: MON-18, MON-30 and MON-82, corresponding to Leishmania donovani sensu stricto, L. infantum and L. archibaldi (still of uncertain taxonomic status), respectively. Southern blotting and PCR 'fingerprinting' revealed identical patterns for all 3 zymodemes, which were indistinguishable from those of L. donovani s.s.
Transactions of the Royal Society of Tropical Medicine and Hygiene 01/1998; 92(1):120-2. · 1.82 Impact Factor
[show abstract][hide abstract] ABSTRACT: The potential use of the recently reported polymerase chain reaction (PCR) protocol for detection of United States epizootic hemorrhagic disease virus (EHDV) serotype 1 (EHDV-1) and serotype 2 (EHDV-2) ribonucleic acid in cell culture and clinical specimens was evaluated for detection of Sudanese EHDV strains. EHDV serotype 5 (EHDV-5) and EHDV, isolate 318 (untyped) designated (EHDV-318), recovered from sentinel calves at the Khartoum University farm (Sudan) were studied. RNA from EHDV-5 and EHDV-318 and a number of EHDV field isolates, propagated in cell cultures, were detected by the described PCR-based assay. The specific 387 bp PCR products were visualized on ethidium-bromide stained agarose gel. Specificity of the PCR products was confirmed by chemiluminescent hybridization with non-radiolabeled internal probe. Amplification product was not detected when the PCR-based assay was applied to RNA from blutongue virus (BTV) prototypes serotypes 2, 10, 11, 13, 16 and 17; total nucleic acid extracts from uninfected BHK-21 cells. The results of this study indicated that the previously described EHDV-PCR assay could be applied for detection of Sudanese as well as United States strains of EHDV serogroup. In addition, the described EHDV-PCR assay could be used as a supportive diagnostic assay to the current conventional virus isolation procedures used for detection of EHDV infection in susceptible ruminants.
[show abstract][hide abstract] ABSTRACT: The performance of the direct agglutination test (DAT) was evaluated under field conditions in an endemic area of visceral leishmaniasis in eastern Sudan, using aqueous (Aq) antigen which has to be kept refrigerated and a newly developed freeze-dried (FD) antigen which is stable at ambient temperature. Both antigens compared well, with 92-98% of readings being identical or only with one dilution difference in titre. FD antigen gave titres that were identical with Aq antigen in 73% of samples, higher in 19%, and lower in 8%. Owing to high ambient temperatures and low humidity, microtitre plate wells dried out during the standard procedures for elution and incubation. However, shortening the elution time from 12 to 4 h proved possible for both antigens; incubation could be reduced from 24 to 10 h for Aq antigen, after which the plates could still be read. Incubation with FD antigen required 18 h and the plates needed to be kept cool because of evaporation. Despite the longer procedure with the FD antigen, the DAT can be completed in 24 h and the use of this stable antigen, that does not require refrigeration, is a major improvement in performing the DAT under unfavourable field conditions.
Transactions of the Royal Society of Tropical Medicine and Hygiene 01/1997; 91(6):671-3. · 1.82 Impact Factor
[show abstract][hide abstract] ABSTRACT: Epizootic hemorrhagic disease virus (EHDV), isolate 318 (EHDV-318), an untyped virus recovered from a sentinel calf herd at the Khartoum University farm in central Sudan, was characterized using molecular biological techniques. With dot blot hybridization technique, a cDNA probe derived from genome segment 6 of EHDV-2 (Alberta strain) hybridized with RNA from EHDV-318. Application of serogroup-specific EHDV polymerase chain reaction (PCR) to EHDV-318 RNA resulted in specific amplification of a 387 bp PCR product. Amplification product was visualized on ethidium bromide-stained agarose gel. Specificity of the PCR products was confirmed by chemiluminescent hybridization with a non-radiolabelled internal probe. No amplification product or hybridization signal was detected when the serotype-specific EHDV-1 or EHDV-2 PCR-based assays were applied to RNA from EHDV-318. The scientific data presented in this study indicated that cDNA probes and serogroup-specific PCR-based assay can be used to classify the virus as a member of EHDV serogroup, and as serotypically distinct from EHDV-1 and EHDV-2.
[show abstract][hide abstract] ABSTRACT: The present work comprises a longitudinal study of Schistosoma mansoni infection in occupationally hyper-exposed canal cleaners in the Sudan and the influence of chemotherapy on humoral immune parameters. The study groups included chronically infected canal cleaners (n = 19), newly recruited canal cleaners (n = 17), normally exposed adults (n = 31), school children (n = 46) and Sudanese negative controls (n = 48). Previous studies of the same canal cleaners have demonstrated that chronically infected canal cleaners were more resistant to reinfection than newly recruited canal cleaners. ELISA was used to detect specific IgE and IgG subclasses in response to whole worm antigen (WWH) and soluble egg antigen (SEA) before and 3 months after praziquantel treatment in the groups of canal cleaners and before and 1 year after treatment in normally exposed adults. When intensity of infection was correlated with IgE antibody response, the resistant group of canal cleaners (those who stopped passing ova after treatment) showed a significant positive correlation between intensity of infection and specific IgE to WWH (Spearman's correlation coefficient = 0.49, P < 0.05) compared with a highly significant negative correlation in the susceptible group (acquired new infection after treatment, Spearman's correlation coefficient = -0.94, P < 0.01). Normally exposed adults and school children had significantly less specific IgE to WWH than canal cleaners, while chronically infected canal cleaners had significantly higher levels of specific IgG1 to WWH than newly recruited canal cleaners and school children, and significantly higher levels of specific IgG4 to WWH than school children. There was a significant increase in specific IgG1 and IgG4 to WWH, 3 months after treatment, in newly recruited canal cleaners and a significant decrease, 1 year after treatment, in normally exposed adults. None of the groups studied after treatment showed a significant change in their specific IgE to WWH. Normally exposed adults had significantly lower levels of specific IgE to SEA than newly recruited canal cleaners, and significantly lower levels of specific IgG1 to SEA than other infected groups. Both newly recruited canal cleaners and school children had significantly higher levels of specific IgG2 to SEA than persons in other groups. Only small differences between groups were observed with regard to specific IgG3 and IgM to SEA. Specific IgG4 to WWH and SEA showed different patterns after treatment between the resistant and susceptible groups of canal cleaners. The resistant group maintained the same level of IgG4 to WWH after treatment compared with a significant increase in the susceptible group. On the other hand, levels of specific IgG4 to SEA showed a highly significant decrease after treatment in the resistant group. In contrast, the same antibody subclass increased after treatment in the susceptible group. Generally, results show an association between IgE and IgG1 responses to WWH and resistance to reinfection. In contrast, an association was observed between IgG2 and IgM responses to SEA and susceptibility to reinfection.
[show abstract][hide abstract] ABSTRACT: The present work was a longitudinal study on Schistosoma mansoni infection in occupationally hyperexposed canal cleaners in the Sudan and the influence of therapy on the parasitological and humoral immune parameters. Chronically infected canal cleaners (n = 28) were more resistant to reinfection (Fisher's exact test, P < 0.05) than newly recruited canal cleaners (n=17). Chronically infected canal cleaners had a significantly higher degree of Symmers' fibrosis (chi2 = 19.1, P < 0.0001), significantly larger portal vein diameter (P < 0.05) and enlarged spleen (chi2 = 4.2, P < 0.05) than recently infected, newly recruited canal cleaners. ELISA was used to detect IgG, IgA and IgM in response to whole worm homogenate (WWH) and cercarial homogenate (CH). Chronically infected canal cleaners had significantly higher IgG to WWH antigen than newly recruited canal cleaners and normally exposed individuals (P < 0.05), while both chronically infected and newly recruited canal cleaners had higher IgG levels to CH antigen than normally exposed individuals (P < 0.05). The newly recruited canal cleaners had a significantly higher IgM level to CH antigen than chronically infected canal cleaners (P < 0.05). The IgG level to WWH antigen increased significantly after treatment in newly recruited canal cleaners and normally exposed individuals (P < 0.05). The IgA level to CH antigen increased significantly after treatment in the chronically infected group (P < 0.05). Comparison of the serological parameters between the different study groups with regards to infection and treatment is discussed.
[show abstract][hide abstract] ABSTRACT: IL-12 is a pluripotent cytokine that interacts with NK and T cells to play a central role in the initiation and maintenance of Th1 responses and IFN-gamma production. Because of the interactive relationship between IL-12 and IFN-gamma response to infectious organisms, a study was undertaken to examine the role of IL-12 in the immune regulation of human visceral leishmaniasis (VL). Human (Hu) VL is associated with immune dysfunction and the appearance of IL-10 mRNA, not present in healed individuals. We found that PBMC from treated VL patients produced both IL-12 p40 and IFN-gamma in response to in vitro stimulation with Leishmania donovani. The production of both IL-12 p40 and IFN-gamma were interdependent and were abrogated by the addition of exogenous Hu rIL-10. In contrast, PBMC from active VL patients did not produce IL-12 p40 or IFN-gamma in response to L. donovani lysate. Neutralizing anti-IL-10 mAb led to the enhancement of IFN-gamma production by active VL PBMC cultured with L. donovani lysate, and this enhanced IFN-gamma production was blocked by anti-IL-12 mAb. The addition of exogenous Hu rIL-12 to PBMC from active VL patients resulted in the augmentation of IFN-gamma in response to L. donovani lysate. Therefore, treatment of active VL patient PBMC with anti-IL-10 or IL-12 shifted the response toward a Th1-type response with the production of IFN-gamma. These results indicate that IL-12 may play an important role in the regulation of the cellular immune responses in Hu VL.
The Journal of Immunology 06/1995; 154(9):4623-9. · 5.52 Impact Factor
[show abstract][hide abstract] ABSTRACT: The main endemic area of kala-azar (visceral leishmaniasis) in the Sudan is in Eastern State and the Blue Nile area of Central State. In order to obtain more recent information about kala-azar in both States, the major hospitals and health centres were visited, the physicians and medical assistants interviewed and available records inspected. In Eastern State a cross-sectional survey of one village was carried out and a longitudinal population-based study of another village started. In this State, after a decline since 1985, a sharp increase in the number of cases was noted from 1991 onwards. This increase was seen in large areas, especially along the Rahad and Dinder Rivers. In contrast, in Central State, there was a decline in the frequency of the disease since the 1960s in the area around Sennar and Singa, which was regarded as a hyperendemic focus up to about 30 years ago. It was hypothesized that this decline may be related to the extensive agricultural development with regular insecticiding and the deforestation of the area. Several aspects with regard to transmission of kala-azar are discussed.
Tropical and geographical medicine 02/1995; 47(4):151-6.
[show abstract][hide abstract] ABSTRACT: The epidemiology, clinical features, pathology, immune responses, diagnosis and treatment of 14 patients with mucosal leishmaniasis in the Sudan are described. The condition occurred mainly in adult males, particularly in certain closely related tribes from the western Sudan. It affected the mucosa of the upper respiratory tract and/or the oral mucosa and sometimes followed treated kala azar. The parasites were sometimes confined to the mucosa, sometimes spread to the lymph nodes, and rarely infected the bone marrow and spleen. One of the 2 patients with both visceral and mucosal leishmaniasis differed from classical kala azar cases; his infection was longer lasting, he was leishmanin positive, and his peripheral mononuclear cells proliferated in response to leishmanial antigens. Mucosal leishmaniasis following treated kala azar is a similar phenomenon to post-kala azar dermal leishmaniasis and post-kala azar uveitis. Post-kala azar mucosal leishmaniasis can therefore be added to the other post-kala azar leishmanial infections. Using the polymerase chain reaction, Southern blot analysis with specific probes, and isoenzyme characterization, the causative parasite was identified as Leishmania donovani in 4 patients and as L. major in one. Unlike American mucocutaneous leishmaniasis, mucosal leishmaniasis in the Sudan was not preceded or accompanied by cutaneous lesions and the response to pentavalent antimony or ketoconazole was good.
Transactions of the Royal Society of Tropical Medicine and Hygiene 01/1995; 89(6):647-52. · 1.82 Impact Factor
[show abstract][hide abstract] ABSTRACT: Between April 1991 and April 1993, a longitudinal study was performed in the village of Um-Salala (1,430 inhabitants) in the endemic area of kala-azar (visceral leishmaniasis) in eastern Sudan. During the two years, a total of 92 kala-azar cases were diagnosed (male:female ratio = 1.8:1, mean age 6.6 years). The annual incidence rates were 38.4/1,000 and 38.5/1,000 person-years, respectively. The ratio of clinical to subclinical cases was 1.6:1 in the first year and 2.4:1 in the second year. Post-kala-azar dermal leishmaniasis occurred in 48 (56%) of 85 kala-azar cases that were followed-up for at least six months. Kala-azar occurred only in previously leishmanin-negative individuals. The majority of the population had a positive leishmanin skin test result, probably due to previous exposure to Leishmania major causing cutaneous leishmaniasis in their homeland in western Sudan from which they had migrated in the 1980s. It was thus postulated that previous cutaneous leishmaniasis might protect against kala-azar but this could not be proved.
The American journal of tropical medicine and hygiene 01/1995; 51(6):826-36. · 2.53 Impact Factor
[show abstract][hide abstract] ABSTRACT: We developed an ELISA test using leishmania antigenic extracts to detect antigen-specific antibody responses, including subclass and isotype analysis, in visceral leishmaniasis (VL) patients from the Sudan. A total of 92 parasitologically proven patients were compared with cutaneous leishmaniasis, schistosomiasis, malaria, onchocerciasis and tuberculosis patients, as well as with healthy endemic and non-endemic controls. Some VL patients were examined before and after chemotherapy. VL patients showed significantly higher IgG responses compared with all other groups (93.4% sensitivity, 93.7% specificity), and higher (but not significantly) IgM responses. All groups showed low IgA levels. All IgG subclasses, IgG1, 2, 3, and 4, showed higher levels in patients than all other groups, with IgG1 and IgG3 levels being significantly reduced following treatment. The rank order for specificity and sensitivity for IgG subclasses was IgG3 > IgG1 > IgG2 > IgG4.