Cryptosporidium and giardia in water in Alexandria: detection and evaluation of viability by flow cytometry and different stains

Cryptosporidium oocysts and Giardia cysts have become ubiquitous in surface waters worldwide. The number and extent of outbreaks of waterborne diseases indicate a significant risk for their possible transmission by drinking-water. Since many Egyptian cities depend on surface water as their main source of drinking water, knowledge of the prevalence of waterborne protozoa in water resources is important. The present study was designed to use flow cytometry to detect Cryptosporidium oocysts and Giardia cysts in water samples in Alexandria city in comparison with the standard staining techniques. Testing the viability of the encountered parasites was also carried out comparing flow cytometry and trypan blue vital stain. Thirty water samples were collected from water tanks from different districts of Alexandria city. Samples were subjected to staining techniques and flow cytometry. Stains used were modified Zeihl-Neelsen [MZN], safranin methylene blue [SMeB], modified trichrome, fluorescent stains [phenol auramine and acridine orange]. Viability was evaluated comparing trypan blue stain and flow cytometry using 4'-6-Diamidino-2-phenylindole immunostain [DAPI]. Flow cytometry proved to be much more sensitive than staining techniques with a sensitivity of 100% for both Cryptosporidium oocysts [30 samples] and Giardia cysts [11 samples]. Following flow cytometry, the fluorescent phenol auramine stain had the greatest sensitivity of 94.74% and 80% [18 and 4 samples, respectively]. The percent of live parasites present in each sample was always significantly higher by DAPI than trypan blue stain. The results of the present study clearly demonstrate that incorporation of flow cytometry can improve sensitivity of detection of Giardia cysts and Cryptosporidium oocysts in water samples. Although it is more expensive than the other staining methods, it is rapid, simple and accurate in estimating the quantity and viability of the parasites in each sample. Thus, flow cytometry can be recommended for detection of protozoa in water

use of ELISA and immunohistochemistry techniques for detection of toxoplasma gondii antigen in tissues of experimentally infected mice

The capability of double antibody sandwich enzyme-linkad immunosorbent assay [ELISA] for detecting antigens of Toxoplasma gondii [T. gondii] in different mice tissue specimens was evaluated in comparison to the immunohistochemistry [IHC] technique. Results proved that tissue antigens were detectable in liver, kidney and mesenteric lymph node [LN] specimens by both methods from the second day of infection, with statistically significant increase in its amount in all organs throughout the period of the study. Using ELISA technique, the highest antigen level was recorded on the second day [0.120 +/- 0.0015] and the fourth day [0.147 +/- 0.0034] of infection in LN specimens, while, the liver showed the highest antigen level at the sixth day post infection [PI][0.165 +/- 0.0066]. On the other hand, using the IHC technique, the highest number of tachyzoites was recorded in LN sections in all studied durations, the second, the fourth and the sixth days PI [1.1 +/- 0.875, 1.6 +/- 1.173 and 3.1 +/- 1.370 respectively]. Thus, sandwich ELISA technique might offer a valuable aid for rapid diagnosis of acute toxoplasmosis in human tissues, and it has proved to be more accurate than IHC technique, since its results was coincided with the pathogenesis of the disease.

Urinary antingen detection for diagnosis of hydatid disease

Hydatid antigen was demonstrated for the first time in urine of patients with hydatidosis by coagglutination test [Co-A]. Urinary antigen was detected in all Co-A positive serum corresponding samples of surgically confirmed hydatid disease. The sensitivity and specificity were 100% in urine compared with the corresponding serum samples. These results clarified that the use of Co-A test for the detection of hydatid antigen in urine is an easy, simple, rapid, noninvasive and efficient method for the diagnosis of hydatidosis

rapid technique for diagnosis of intestinal parasitic infection

This study was carried out on stool specimens of 150 cases attending the outpatient clinics of the Faculty of Medicine, Alexandria University. All patients were suffering from gastro-intestinal disturbance specially diarrhoea. Each specimen was examined for intestinal parasites by direct saline and iodine smears and was stained by using: quantitative buffy coat [QBC] tube technique, modified trichrome and modified Ziehl Neelsen stains. This work aimed to evaluate the quantitative buffy coat tube method in diagnosis of parasitic infection in such cases, comparing it with the other staining reference techniques used simultaneously. Out of 150 diarrhaeic patients 67 were positive for protozoa. These protozoa identified in the examined specimens were Giardia lamblia, Cryptosporidium parvum, Cyclospora, Microsporidia and Blastocystis hominis. The results showed high sensitivity and specificity [100%] of the QBC tube method over the other staining techniques

In-pouch TV culture system in diagnosis of trichomonas vaginalis infection

A newly simplified culture method, the In-Pouch TV culture system, the wet mount [WM] examination and the acridine orange stain were compared with the Oxoid culture as a standard technique in the diagnosis of trichomoniasis. Out of 70 symptomatic cases enrolled in this study, 28 specimens were positive by all methods. Among these positive specimens, 21 were positive by the Oxoid culture [75%] and 24 [85.7%] with sensitivity of 85.7% by the In-Pouch system. Both wet mount preparation and acridine orange stain had less sensitivity than the In-Pouch system [61.9%] and detected 15[53.6%] and 16 [57.1%] of the cases respectively. The In-Pouch system has been proved to be easier in the transport and culture technique than the ordinary culture method. It alleviates the need to enter the culture, thus prevents contamination. Its cost is comparable to the ordinary culture tube. Therefore, it is recommended to use the In-Pouch culture system as a method of diagnosing trichomoniasis

Genetical and electron microscopical studies on cryptosporidia

The present work was to clarify whether C. parvum oocysts have different strains in human patients by using different staining, electron microscopical and genetical techniques. A trial to induce a vaccine against Cryptosporidial infection in mice was carried out using killed autoclaved Cryptosporidial oocysts. The results obtained were satisfactory. Two genotypes of C. parvum. viz: human and zoonotic genotypes were detected and described. The killed vaccine used orally gave promising results

Diagnosis of blastocystis hominis by different staining techniques

150 stool samples were collected from diarrheic patients of different ages and examined for Blastocystis hominis by direct smears and concentrated by Sheather's sugar flotation. Staining was done by Giemsa, 2 modifications of trichrome stain, modified Ziehl-Neelsen, Safranin-methylene blue and 2-auramine stains. Out of the 150 cases, 9 were positive for blastocystosis. The best stains were Safranin- methylene blue and modified Ziehl-Neelsen stains. They had the advantage of staining cysts and amoeboid forms besides being rapid and easy to perform. The modified trichrome stains were less specific and time-consuming. Giemsa stain was not an efficient stain. Scanning and transmission electron microscopy [SEM and TEM] were performed to study the fine ultrastructure

Use of lactoferrin assay in the diagnosis of different vaginal pathogens

Local cell mediated immunity is more important than systemic immunity for protection against different vaginal pathogens. The predominant inflammatory cells in vaginal pool are almost exclusively polymorphonuclear neutrophils [PMN] which were varied according to the type of vaginal pathogens, lactoferrin is an iron binding glycoprotein found in the secondary granules of PMN. In order to determine the usefulness of such marker for neutrophilic activity in different vaginal infections, we examined the vaginal discharge using antilactoferrin antibodies [lactoferrin latex agglutination test: LFLA].Against different gold standard techniques. Our results demonstrated that Trichomonas vaginalis [T.V] revealed a high lactoferrin titer which was positively correlated with the number of PMN. In addition, cases with vaginal candidiasis [V.C] was characterized by mild inflammatory process as expressed by mild lactoferrin level which was also correlated with the PMN count. However, a paradoxic finding was observed in discharge recovered from cases with bacterial vaginosis [B.V] where lactoferrin titer was not correlated with PMN count. In addition, the findings of the present work indicated that LFLA was sensitive and specific when used alone and its sensitivity was increased after coupling with other simple methods as pH determination or amin test. In conclusion, our results described the feasibility of using LFLA as a simple, extremely sensitive, reliable method in distinguishing different types of vaginal pathogens, so it could be used as a promising method for a widespread community screening to diagnose population of females at risk