Preparation of a K39sub recombinant antigen for the detection of Leishmania infantum antibodies in human: a comparative study with an immunochromatographic test and direct agglutination

The Mediterranean type of kala-azar is occurred in different parts of Iran and caused by Leishmania infantum. A rapid and valid test for early detection of visceral leishmaniasis in human would be highly desirable because it could decrease mortality rate of the disease. In this study, we aimed to compare the results of K39sub antigen with a commercial immunochromatographic dipstick rk39 test [Cypress Diagnostic Company, Belgium] for early detection of L. infantum infection in human. K39sub recombinant antigen of L. infantum LON49 was expressed in prokaryotic system and evaluated for the diagnosis of human visceral leishmaniasis. This study evaluated the performance of recombinant K39sub antigen by ELISA and an commercial immunochromatographic dipstick rk39 test for the detection of L. infantum antibodies in 43 clinically infected patients with direct agglutination test [DAT] at a 1: 3200 cut off titer and higher. Controls included 69 healthy volunteers and 28 patients with other diseases including malaria [n=5], tuberculosis [n= 3], toxoplasmosis [n= 4], cystic hydatidosis [n= 5] and cutaneous leishmaniasis [n= 11]. The sensitivity of the K39sub antigen and an immunochromatographic dipstick rk39 test was 90.7%, and 97.7%, respectively, while the specificity was 95.6% and 97.9%, correspondingly. A good concordance was found between k39sub antigen and commercial dipstick rk39 strips [k= 96.4%]. The accuracy of the K39sub antigen in the detection of L. infantum antibodies in human infection is confirmed

Diagnosis of canine visceral leishmaniasis by ELISA using K39sub recombinant antigen

Surveillance of the canine reservoir is highly important to help control of visceral leishmaniasis in human. It is therefore imperative to improve and develop new tools reliable, easy to use, and cheap for the diagnosis of canine leishmaniasis. K39 sub recombinant antigen of Leishmania infantum was expressed in prokaryotic system and evaluated for sero-diagnosis of canine visceral leishmaniasis [CVL]. The gene fragment encoding a single 39-amino acid subunit of the kinesin-related protein k39 [k39sub] was amplified from DNA of Iranian strain of L. infantum [MCAN/IR/96/LON49] and cloned into a pMAL-p2 expression vector in frame with maltose-binding protein [MBP] fusion. The antigenic properties of L. infantum recombinant K39 subunit [39 amino acids] have been tested for the serological diagnosis of CVL by ELISA. K39sub ELISA for CVL was compared with a standard direct agglutination test [DAT] on 55 clinically infected dogs and 71 healthy controls from endemic areas of Ardabil and East Azerbaijan provinces, north-west of Iran. A sensitivity of 72.7% and specificity of 87.3% were found at a 1:320 cut off titer when DAT confirmed cases were compared with healthy control. A good concordance was found between k39sub ELISA and DAT [k= 81.0]. Given the antigenic properties shown by the k39sub, we think this protein carry immunodominant epitopes and are valuable for the sero- diagnosis of L. infantum infection in dogs

[Evaluating the presence of testis specific transcripts in mature human spermatozoa]

Progress and completion of spermatogenesis is related to simultaneous expression of various genes. Recent studies show that many genes are expressed in the sperm and several RNA copies are present in the mature spermatozoa. Identification of these genes and evaluation of their functions would improve our understanding of the molecular basis of fertilization, early embryo cleavage and the causes of many types of unexplained male infertility. In this study, we investigated the expression of DAZ, PRM1, PRM2, TSGA10, SYCP3 and AKAP4 genes in ejaculated human spermatozoa. Semen samples were collected from men referring to Avicenna Infertility Clinic. Normal semen samples [According to WHO criteria] were subjected to density-gradient centrifugation to specifically recover the pure fraction of motile spermatozoa with normal morphology. Total RNA was extracted from sperm pellets and cDNA was synthesized using RT-PCR. The presence of DAZ, TSGA10, PRM1 and PRM2 cDNAs were evaluated using appropriate primers. Expression of SYCP3 [Testis specific gene] was evaluated by nested RT-PCR. The cDNA synthesized from normal testis tissues was used as positive control. Study on cDNAs showed that DAZ, TSGA10, PRM1 and PRM2 transcripts were present in normal human testis and all of the evaluated mature spermatozoa samples but not AKAP4 or SYCP3 transcripts. According to our previous study, the expression of SYCP3 and AKAP4 genes is started from spermatocyte level in human testis during spermatogenesis process. However, we did not found any transcripts of these genes in mature spermatozoa. It is estimated that mRNAs of TSGA10, PRM1, PRM2 and DAZ and other testis specific genes in spermatozoa may participate in later sperm functions such as fertilization and early embryo cleavage. Therefore, further studies are needed to understand the role of these transcripts in the process of fertilization and early embryo development

Polymorphism in phospholipid hydroperoxide glutathione peroxidase [PHGPX] gene in Iranian infertile men

Leukocytes and defective or dead spermatozoa in human semen are a source for the production of reactive oxygen species [ROS] and subsequent injury to intact sperms. Enzymatic and non-enzymatic defensive mechanisms in semen detoxify these compounds. Glutathione peroxidase-4 [GPX-4 or PHGPX] is a major selenoprotein in sperm and it is one of the enzymatic mechanisms that play multiple roles during spermatogenesis. Some of these roles are formation of the mitochondrial capsule, hydroperoxide detoxification and sperm chromatin condensation. Any decrease in the enzyme activity or content, may create disorders in spermatogenesis and sperm fertilizing ability. Considering defects in the expression of the enzyme gene or presence of mutations which may cause decreases in PHGPX activity or content, this study was carried out to identify a number of important mutations in GPX-4 gene by PCR-RFLP method in Iranian infertile men. This study was performed on 128 Iranian men who had been referred to Avesina Infertility Clinic, including 74 infertile men with defective sperm parameters, 18 normozoospermic and 36 fertile subjects as controls. Mean +/- SD for sperm parameters were determined. Genomic DNA was extracted using salting out procedure from peripheral blood leukocytes. PCR-RFLP was done by two sets of primers with 237 bp and 148 bp PCR products that were designed for 1A and 4 exons of GPX-4 gene covering nucleotides of+6 [C[right wards arrow]T], +17 [G[right wards arrow]A], +1725 [G[right wards arrow]A] by Mwol, PshAI and SatI enzymes. Digestion of a 237 bp intact PCR product by Mwol generates two fragments [151 bp and 86 bp]. When a mutation occurs in the restriction site +6 [C[right wards arrow]T], the enzyme would not recognize the sequence, therefore 237 bp segment remains undigested. Treatment of 237 bp segment with PshAI generates two fragments [161 bp and 76] in the intact gene but the same enzyme can not digest 237 bp segment when a mutation occurs in the restriction site +17 [G [right wards arrow] A]. Ultimately, digestion of 148 bp intact segment with SatI generates two fragments [108 bp and 40 bp] but when a mutation occurs in the restriction site +1725 [G [right wards arrow] A], the enzyme will not recognize the sequence; therefore 148 bp segment remains undigested. Enzymatic digestion evaluations of 237 bp and 148 bp segments in all participants revealed that neither of the examined mutations existed in GPX-4 gene. According to the results of this study, it is determined that the prevalence of these mutations in Iranian infertile men is probably low and it may have no association with the etiology of the disorder affecting sperm parameters. Hence, a study with a larger number of patients is suggested to determine the exact prevalence of these and other mutations of the gene in Iranian infertile men

Effect of post ingestion and physical conditions on PCR amplification of host blood meal DNA in mosquitoes

Identification of host blood meal in haematophagous arthropods is an important element in their rule in transmission of vector borne diseases. The effects of post ingestion and physical conditions that killed mosquitoes are stored on the success of detecting blood meal DNA of Anopheles stephensi and Culex quinquefasiatus was investigated by polymerase chain reaction [PCR] amplification at the human mitochondrial DNA cytochromeB [CytB] gene. Host DNA extracted from the blood meal up to 33 h post ingestion in both species acts as an efficient template for PCR amplification. However more DNA concentration needs for meals digested longer time. Successful PCR amplification among meals digested for 36 h dropping to a faint band. There were no differences between PCR success rate for sampled stored at +4°C or -20°C, but less successful products were observed in samples kept at 4°C for periods longer than 30 h digestion. The results of this study is important in malaria epidemiological studies to provide valuable information about the degree of contact between human hosts and mosquito vectors, impact of vectors controls such bed nets and repellents, and the transmission dynamics of human malaria and other vector-borne diseases