Ancylostoma duodenale infection: a study of serum immunoglobulin G4 response to the excretory secretory antigen of adult worm.

The serum anti-Ancylostoma duodenale immunoglobulin (Ig) G4 antibody response to fraction III of the partially purified excretory secretory antigen of adult worm (Ad III ESA) was studied. The work included 60 patients with A. duodenale infection (GI), 40 patients with other parasitic infections (GII) and 30 apparently healthy parasite-free controls (GIII). Level of serum specific IgG4 was measured by indirect enzyme linked immunosorbent assay and compared with serum specific IgG, IgG 1, 2 & 3 subclass antibodies. Patients of GI had gastro-intestinal manifestations and symptoms suggestive of anaemia, and by investigations they had anaemia in 31.7% & eosinophilia in 100%. Measuring the intensity of A. duodenale infection, quantified as fecal egg counts, in patients of GI revealed that 60%, 30% & 10% had light, moderate, and heavy infections, respectively. The serum anti-Ad III ESA IgG & IgG 1-4 subclass antibodies were significantly elevated (P < 0.001) in patients of GI compared with GIII. Serum specific IgG4 was expressed in 100% of patients of GI at a significantly highly elevated level than IgGI (P < 0.01), IgG2 & IgG3 (P < 0.001). Specific IgG1 was expressed in 88.3% of patients of GI at a significantly elevated level (P < 0.001) than IgG2 & IgG3 which were expressed in 31.7% & 38.3%, respectively and elevated to a moderate extent. Serum specific IgG4 showed a 1.0, 1.1, 3.1 & 2.6-fold increase in detection rate of positive cases than IgG, IgG1, IgG2 & IgG3, respectively. The highest ability to differentiate between infected and healthy subjects was by serum specific IgG4 recording a discrimination coefficient of 9.4, while IgG, IgG1, IgG2 & IgG3 recorded 5.2, 6.3, 3.2 & 3.4, respectively. Serum specific IgG4 showed a significant positive correlation (r = 0.41, P < 0.001) with the intensity of A. duodenale infection, as was demonstrated by IgG & IgG3 (P < 0.01 & P < 0.05, respectively). Detection of serum anti-Ad III ESA IgG4 antibody recorded a 100% sensitivity that was significantly higher (P < 0.001) than IgG1, IgG2 & IgG3, but insignificantly different (P > 0.05) from IgG. Finally, serum specific IgG4 recorded a 100% specificity that was significantly higher than IgG, IgG2, IgG3 (P < 0.01) & IgG1 (P < 0.05). They showed cross-reactions with ascariasis, lymphatic filariasis and strongyloidiasis. The results are discussed.

Human giardiasis as an etiology of skin allergy: the role of adhesion molecules and interleukin-6.

The role of adhesion molecules; the intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) as mediators in development of skin allergy caused by giardiasis and the controlling role of the cytokine interleukin (IL)-6 over these adhesion molecules were studied. The work included 25 symptomatic giardiasis patients with skin allergy manifested by diffuse urticaria, pruritus, wheal and erythema, and had positive serum anti-Giardia immunoglobulin (Ig) E measured as mean optical density (OD) value by enzyme linked immunosorbent assay (ELISA), employed as an evidence of allergic sensitization (G.I). They were compared with 30 symptomatic giardiasis patients (G.II) and 20 apparently healthy control subjects (G.III), both latter groups had negative serum anti-Giardia IgE. The mean OD value of anti-Giardia IgE was significantly increased in G.I (P < 0.01) & insignificantly different in GIII (P > 0.05) compared with G.III. Serum levels of soluble forms of adhesion molecules; sICAM-1 & sVCAM-1, and IL-6 were determined by ELISA. sICAM-1 & sVCAM-1 serum levels were significantly increased (P < 0.001) in G.I compared with G.III and showed insignificant difference (P > 0.05) between Gs. II & III. Serum IL-6 significantly increased in G.I (P < 0.001) & G.II (P < 0.05) compared with G.III, and was significantly higher (P < 0.001) in G.I than G.II. Serum IL-6 correlated positively with serum sICAM-1 (P < 0.01) and sVCAM-1 (P < 0.001) in G.I. The results are discussed.

Alkaline phosphatase from Echinococcus granulosus metacestodes for immunodiagnosis of human cystic echinococcosis.

The work examined the use of Echinococcus granulosus alkaline phosphatase (EgAP) (extracted from hydatid cyst membranes) as an antigen for immunodiagnosis of human cystic echinococcosis (CE). It was assessed by enzyme-linked immunosorbent assay (ELISA) and Western immunoblotting (IB) for detection of serum anti-EgAP immunoglobulin (Ig)G antibody and was compared with hydatid cyst fluid (HCF). The EgAP and HCF were of sheep liver cysts origin. Sera from 30 patients with surgically confirmed CE (G. I), 30 patients with other parasitic infections (G. II), and 20 healthy controls (G. II) were examined. The mean optical density of each of anti-EgAP IgG and anti-HCF IgG antibodies in G. I was significantly higher (P < 0.01) than in each of G. II and III. The use of EgAP in ELISA showed 100% sensitivity and specificity recording significantly higher sensitivity (P < 0.05) and specificity (P < 0.01) than when using HCF in ELISA which showed 86.7% sensitivity and 84% specificity. SDS-PAGE resolution, under reducing conditions, of EgAP revealed a molecular weight of 56 KDa, while that of HCF revealed a number of antigenic bands ranged from 12 to 130 KDa. IB analysis showed that sera from CE patients recognized the EgAP 56 KDa and also one or more of HCF antigenic bands of molecular weights at 116, 63, 44, 39, 24, 20, 16 and 12 KDa. The use of EgAP in IB showed 100% sensitivity and specificity recording an insignificant difference (P > 0.05) in sensitivity and a significantly (P < 0.05) higher specificity than when using HCF in IB which showed 100% sensitivity and 90% specificity. Cross reactivity with HCF in ELISA and IB was seen with schistosomiasis mansoni, fascioliasis, hymenolepiasis nana and ascariasis. Using EgAP, there was an insignificant difference (P > 0.05) in each of the sensitivity and specificity between ELISA and IB. Using HCF, there was a significantly (P < 0.05) higher sensitivity and an insignificantly (P > 0.05) higher specificity by IB than ELISA. The implications of these results are discussed.

Pulmonary trichomoniasis: improved diagnosis by using polymerase chain reaction targeting Trichomonas tenax 18S rRNA gene in sputum specimens.

A polymerase chain reaction (PCR) assay targeting species-specific region in 18 small subunit ribosomal RNA gene of Trichomonas tenax was used to examine sputum specimens in order to diagnose pulmonary trichomoniasis caused by T. tenax. It was compared with wet mount preparation, Giemsa-stained smear, and Kupferberg Trichononas broth culture for detection of T. tenax trophozoites in sputum. The study included 250 individuals; 100 immunocompromised patients with chest complaints (group I) and 100 patients with chronic pulmonary diseases (group II), and 50 healthy individuals as controls (group III). 20 cases among all examined were positive in one or more method giving for pulmonary trichomniasis a total prevalence of 8%; 12 cases (12%) in group I, 8 cases (8%) in group II, and none in group III, with no significant difference between groups I & II. Pulmonary trichomoniasis was prevalent at age ranged between 31 to 50 years, and in total males (10%) than females (5.5%) with no significant difference. Among the 200 examined patients, pulmonary trichomoniasis had a prevalence of 3% by wet mount, 2.5% by Giemsa-stained smear, 7% by culture, compared to 10% by PCR. Culture was used as reference standard. All culture positive specimens were PCR positive showing a product at 0.8 Kb long by agarose gel electrophoresis, and giving a 100% sensitivity. Wet mount, Giemsa-stained smear, and culture had a sensitivity of 43%, 35.7%, and 70%, respectively. No PCR negative specimens were positive by any of the other methods. 6 specimens were culture negative PCR positive and remained PCR positive when retested 3 times. The calculated specificity of PCR was 97%. NO PCR target product was amplified with DNAs of T. vaginalis and various pulmonary pathogens. The results are discussed.

Secretory and humoral antibody responses to Blastocystis hominis in symptomatic and asymptomatic human infections.

The study included 3 groups of individuals, in the first 2 groups they had positive stool microscopic examinations only for B. hominis indicating blastocystosis, with and without gastrointestinal symptoms, respectively, while the last group included apparently healthy individuals with no parasites in stool. Stool and serum samples of these individuals were subjected to detection of anti-B. hominis fecal and serum IgA and serum IgG antibodies by indirect ELISA, and detection of B. hominis fecal and serum antigens by double sandwich ELISA. In symptomatic B. hominis infections with positive stool microscopy the study recorded first: specific secretory IgA and humoral IgA and IgG antibody responses at a prevalence of 100%, 83.3% and 86.6%, respectively, with an increased significant difference (P<0.001) of each from healthy controls, together with an increase in level of secretory IgA than that of humoral IgA antibody (P<0.001), and second: the presence of specific antigens in stool and serum at a prevalence of 96.6% and 90%, respectively. With an increased significant difference (P<0.001) of each from healthy controls together with the former at a higher level than the latter (P<0.05). In asymptomatic B. hominis infections with positive stool microscopy the study recorded first; absence of each of the studied specific secretory and humoral antibody responses with no significant difference (P>0.05) of each from healthy controls, and second; absence of specific antigens in stool and serum with no significant difference (P>0.05) of each from healthy controls nor from each other. The explanations and implications of these results are discussed.

Cystatin capture-dot-enzyme-linked immunosorbent assay for immunodiagnosis and assessment of cure of experimental trichinellosis in mice.

Cystatin capture dot enzyme-linked immunosorbent assay (CC-dot ELISA) was evaluated as a new version of ELISA for diagnosis of trichinellosis by the detection of anti-Trichinella spiralis cysteine proteinase (CyP) IgG, using nitrocellulose membrane sensitized with cystatin as a capture reagent for CyP from T. spiralis muscle larvae excretory secretory products (ESP) without purification, compared to the detection of anti-T. spiralis IgG by conventional (conv.) ELISA, using whole ESP. Experimentally infected mice with light (100 larvae/mouse) and heavy (300 larvae/mouse) infections by T. spiralis larvae at 7, 14, 21 and 56 days post infection, and after flubendazole treatment were examined. As early as one week post infection CC-dot ELISA gave high positive rates of 86.6% and 100% in light and heavy infections, respectively, in contrast to negative results by conv. ELISA. CC-dot ELISA showed in light and heavy trichinellosis a higher efficiency in comparison to conv. ELISA (97.7% versus 67.7% and 98.8% versus 80%, respectively) and a higher overall sensitivity (96.6% versus 55% and 98.3% versus 73.3%, respectively). No cross reactions with sera of other parasitic infected or non infected control mice were recorded by CC-dot ELISA giving 100% specificity compared to 93.3% by conv. ELISA. After treatment, CC-dot ELISA gave positive results only in uncured mice with remaining muscle larvae, while conv. ELISA was positive in mice with and without remaining muscle larvae. CC-dot ELISA used lower quantities of antigen, was performed at room temperature, and read by naked eye in less than 2.5 hours.

Role of nitric oxide in host defense against Hymenolepis nana infection.

The defensive role of nitric oxide (NO) against Hymenolepis nana was investigated in vivo and in vitro studies. Serum NO levels were increased (P < 0.001) in mice 5 days (cysticercoid stage) and 15 days (adult stage) after H. nana induced oral infection with 1000 eggs/mouse, compared with normal controls. Meanwhile, L-NAME, a NO synthase inhibitor, oral administration in drinking water to infected mice caused a non significant decrease in serum NO levels (P > 0.05) compared with normal controls, and was associated with a significant increase in number of both cysticercoids and adult worms (P < 0.001) compared to that in infected mice 5 and 15 days post infection. In an in vitro study, the NO donor; sodium nitroprusside caused an increased mortality rate of H. nana cysticercoids and adult worms (P < 0.001) compared with controls without NO donor, and this was in a concentration-dependent manner (P < 0.001). Implications of these results are discussed.

Detection of Toxoplasma gondii antigens in urine by reverse latex agglutination test during human toxoplasmosis.

T. gondii antigens were detected in concentrated urine samples of 39 out of 40 patients with acute toxoplasmosis and in 38 serum samples of the same patients by the reverse latex agglutination (RLA) test which showed a high sensitivity of 97.5% and 95%, respectively. By capture enzyme-linked immunosorbent assay (ELISA). T. gondii antigens were detected in concentrated urine samples of 30 out of the 40 patients in 29 sera of the same patients, recording a moderate sensitivity of 75% and 72.5%, respectively. T. gondii antigens were not detected in urine or serum samples of patients with chronic toxoplasmosis by neither RLA test nor ELISA. No false positive reaction was observed with urine or serum samples of normal--or other parasitic infection--control individuals, recording a specificity of 100% for each of RLA test and ELISA. Urine samples were collected easily and readily without causing any inconvenience to the individuals enrolled in the study. The RLA test was easy to perform and visually interpreted within 2-3 minutes. The diagnosis of acute toxoplasmosis through the detection of easily obtainable urinary T. gondii antigens by the highly sensitive and specific RLA test is discussed.

The use of Ziehl-Neelsen stain, enzyme-linked immunosorbent assay and nested polymerase chain reaction in diagnosis of cryptosporidiosis in immuno-competent, -compromised patients.

In the present study, Cryptosporidium parvum was diagnosed in stool by Z-N stain, ELISA and PCR. The detected cases were 5.3%, 8.3% and 9.6% by the previous three methods, respectively. The sensitivity, specificity and accuracy of the different techniques were evaluated. The Z-N stain showed the lowest sensitivity and accuracy in relation to either ELISA or PCR. Moreover, the study revealed that the sensitivity, specificity and accuracy of ELISA detection of Cryptosporidium in relation to detection of DNA in stool by PCR were 84.2%, 96% and 88.8%, respectively. Consequently, PCR showed the best results. From a practical point of view, ELISA is recommended for wide spread use in diagnosis of cryptosporidiosis.

A preliminary study on the relationship between Trichomonas vaginalis and cervical cancer in Egyptian women.

The relationship between Trichomonas vaginalis and cancer cervix was investigated by detection of T. vaginalis antibodies, in the sera of 48 invasive cervical cancer patients and 100 random age matched female control, using western immunoblot technique. It was found that antibodies to T. vaginalis were detected in sera of 18.75% (9/48) of cervical cancer patients compared with 5% (5/100) of controls. The increase was evident in age group, 40-49 years (21.05% vs 5%) and of those with squamous cell carcinoma (6/9) and mainly with grade II & III. All the reactive sera of invasive cancer patients reacted strongly with T. vaginalis surface antigen of about (109.9, 86.1, 56.2, 48.2 and 30 Kda). So, there may be an association between T. vaginalis and the risk of cervical cancer, as there was more than 3 fold increase in the prevalence of T. vaginalis antibodies in patients with invasive cervical cancer compared to age matched female controls. This study highlights the importance of clinically detection of T. vaginalis infection, which is one of the group of factors involved in the genesis and progression of cervical cancer. In addition, its treatment would aid in restricting the rising incidence of this dreaded disease.

Effect of the iron chelator deferoxamine on Trichomonas vaginalis in vitro.

The effect of 10 microMol, 15 microMol, 30 microMol, and 60 microMol concentrations of deferoxamine (DFO), a clinically approved iron chelator, was determined on viability and multiplication of Trichomonas vaginalis grown in TYM axenic culture medium at 24 hours interval. DFO killed all T. vaginalis isolates with a minimum lethal concentration of 30 microMol after 48 hours culture incubation with the drug. A potent and persistent inhibitory effect of DFO on the parasite viability and multiplication was recorded throughout the study till its end, in a drug concentration and time exposure-dependent manner. Furthermore, the present work studied the proteinase activity of T. vaginalis grown for 48 hours in DFO inoculated TYM medium, and recorded a significant decrease by all drug concentrations applied in the work. Different possible mechanisms of action of DFO against T. vaginalis and its possible use for treatment of trichomoniasis are discussed.