257
Total
DNA probe to detect Giardia lamblia
genomic
SIR,-Whereas the development of cloned DNA probes for the detection of parasites’ is difficult, total genomic DNA probes are simple to produce and may give comparable sensitivity and specificity when used under controlled hybridisation conditions. 2-4 We report a radiolabelled total genomic DNA probe to detect Giardia lamblia trophozoites and cysts which shows similar specificity and sensitivity to a cloned probe reported previously.5 The probe was prepared by purifying and radiolabelling total G lamblia genomic DNA, as described previously for Trypanosoma cruzi.3 Samples (extracted DNA, trophozoites, or cysts) were spotted directly onto a nylon filter, denatured in alkali, and covalently linked to the filter by ultraviolet illumination. Cysts were exposed to 1-7 mol/I guanidine thiocyanate for 25 minutes at room temperature before spotting, since this enhanced hybridisation significantly. Prehybridisation for 2-4 h at 45°C in a solution of 50% formamide, 6 x saline sodium citrate (SSC), 0-05 mol/I sodium phosphate buffer pH 66, 5 x Denhardt’s solution, and salmon sperm DNA, was followed by hybridisation with the denatured probe for 16-20 h at 42°C in a solution of 6 x SSC, 0-01 mol/I sodium phosphate buffer pH 66, 1 x Denhardt’s solution, 50% formamide, 10% dextran sulphate, and salmon sperm DNA. Unbound probe was removed by high stringency washes before autoradiography. Using this probe, we obtained a hybridisation signal with 10 ng of G lamblia DNA, 105 trophozoites, and 1()4 guanidine-thiocyanate-treated cysts (figure). This compares favourably with a sensitivity of about 105 cysts using a radiolabelled probe selected from a cloned Giardia DNA library.5 There was no cross-reaction with the closely related Trichomonas vaginalis or with Entamoeba histolytica (figure), or with Crithidia fasciculata or Leishmania donovani. Though the probe cross-hybridised with
05 µg of T cruzi DNA, this would not be a problem in practice, and at 50 µg the amount of mammalian DNA needed to give a signal was orders of magnitude greater than the amount of Giardia DNA. The microscopic detection of G lamblia cysts in faecal smears may be only about 50% successful,6 and cysts present in water may be missed at concentrations below 4000/1.7 Thus, DNA probes may offer advantages over traditional methods of detecting Giardia. With the introduction of non-isotopic labels we would advocate their wider use as diagnostic tools. Division of Communicable Diseases, St George’s Hospital Medical School, London SW17 ORE, UK
Department of Medical Protozoology, London School of Hygiene and Tropical Department of Pure & Applied Biology, Imperial College of Science, Technology & Medicine, London
D. J. M. LEWIS Medicine
E. L. GREEN
F. ASHALL
1. McManus DP, Simpson AJG. Identification of Echinococcus organisms using cloned DNA markers. Mol Biochem Parasitol 1985; 17: 171-78. 2. Greig S, Ashall F. Detection of South American trypanosomes in insects using total parasite DNA probes. Parasitol Today 1987; 3: 375-76. 3. Ashall F, Yip-Chuck DAM, Luquetti AA, Miles MA. Radiolabelled total parasite DNA probe specifically detects Trypanosoma cruzi in mammalian blood. J Clin Microbiol 1988; 26: 576-78. 4. Flisser A, Reid A, Garcia-Zepeda A, McManus DP. Specific detection of Taenia saginata eggs by DNA hybridisation. Lancet 1988; ii. 1429-30. 5. Butcher PD, Farthing MJG. DNA probes for the faecal diagnosis of Giardia lamblia infections in man. Biochem Soc Trans 1989; 17: 363-64. 6. Kamath KR, Murugasu R. A comparative study of four methods for detecting Giardia lamblia in children with diarrhoeal disease and malabsorption. Gastroenterology 1974; 66: 16-21. 7. Feachem RG, Bradley DJ, Garelick H, Mara DD. In: Sanitation & disease: health aspects of excreta and waste water management. London: John Wiley & Sons, 1983: 349-56.
Normal
development after exposure to mifepristone in early pregnancy
Results of hybridisation with the total genomic Giardia DNA
probe. 1 =Glamblia DNA 1.5 J.!g (A), 05 g (B), 0.1 J.!g (C), 10 ng (D), 1 ng (E), 2=vaginalis DNA 5 pg (A), 0 5 µg (B), 50 ng (C); 3=Ehistolytica trophozoites 6 x 105 (A), 6 x 10’ (B); 4=T cruzi DNA 5 µg (A), 0,5 µg (B), 50 ng (C), 5=rat DNA 5 wg (A), 0-5 pg (B), 50 ng (C); 6=G lamblia trophozoites 10. (A), 5x 105 (B), 105 (C), 10’ (D), 103 (E), 500 (F); 7 = G lamblia cysts 105 (A), 6x10" (B), 310" (C), 10"(D), 6x 103 (E)
SIR,-Mifepristone (RU38486), due to its ability to block the action of progesterone in the pregnant uterus, offers a novel approach to drug-induced termination of pregnancy.’ Given alone to terminate early pregnancy mifepristone is effective in about 80% of cases. The subsequent administration of subtherapeutic doses of a prostaglandin analogue increases efficacy to 95%. It can also be given alone as a cervical ripening agent before transcervical operative procedures. Mifepristone has teratogenic effects in the rabbit but not in other speciesand it may have been associated with a malformed fetus aborted in the second trimester after the mother took the drug to terminate an early pregnancy.3 We wish to report the outcome of the pregnancies of three women who decided to continue to term, despite having taken mifepristone during participation in one of two clinical trials. Cases 1 and 2 were recruited into a study of mifepristone and gemeprost in the termination of early pregnancy. Case 3 took part in a double-blind, placebo-controlled trial of the cervical-priming properties of mifepristone before surgical termination of pregnancy. All patients gave written informed consent. Case 1. A 21-year-old primigravida was given mifepristone at 8 weeks’ gestation. Before prostaglandin administration, the patient indicated that she did not wish to proceed with the termination. Throughout the pregnancy, ultrasound scans showed satisfactory fetal growth. The patient reported two episodes of light brown vaginal discharge. The pregnancy progressed satisfactorily, and the patient went into spontaneous labour at 40 weeks’ gestation and delivered a healthy boy weighing 4150 g. Postnatal examination of the infant revealed no abnormality and he continues to develop normally 15 months later. Case 2. A 31-year-old (para 2 + 0) was given mifepristone at 8 weeks’ gestation. Before administration of the prostaglandin she indicated that she did not wish to proceed with the termination. 2 days later the patient was admitted with vaginal bleeding and mild pelvic cramps. Bleeding settled after 3 days in hospital and ultrasound scan confirmed a viable pregnancy. Subsequent antental progress was uncomplicated. The patient was admitted in labour at 39 weeks’ gestation and delivered a healthy male infant weighing
Total
DNA probe to detect Giardia lamblia
genomic
SIR,-Whereas the development of cloned DNA probes for the detection of parasites’ is difficult, total genomic DNA probes are simple to produce and may give comparable sensitivity and specificity when used under controlled hybridisation conditions. 2-4 We report a radiolabelled total genomic DNA probe to detect Giardia lamblia trophozoites and cysts which shows similar specificity and sensitivity to a cloned probe reported previously.5 The probe was prepared by purifying and radiolabelling total G lamblia genomic DNA, as described previously for Trypanosoma cruzi.3 Samples (extracted DNA, trophozoites, or cysts) were spotted directly onto a nylon filter, denatured in alkali, and covalently linked to the filter by ultraviolet illumination. Cysts were exposed to 1-7 mol/I guanidine thiocyanate for 25 minutes at room temperature before spotting, since this enhanced hybridisation significantly. Prehybridisation for 2-4 h at 45°C in a solution of 50% formamide, 6 x saline sodium citrate (SSC), 0-05 mol/I sodium phosphate buffer pH 66, 5 x Denhardt’s solution, and salmon sperm DNA, was followed by hybridisation with the denatured probe for 16-20 h at 42°C in a solution of 6 x SSC, 0-01 mol/I sodium phosphate buffer pH 66, 1 x Denhardt’s solution, 50% formamide, 10% dextran sulphate, and salmon sperm DNA. Unbound probe was removed by high stringency washes before autoradiography. Using this probe, we obtained a hybridisation signal with 10 ng of G lamblia DNA, 105 trophozoites, and 1()4 guanidine-thiocyanate-treated cysts (figure). This compares favourably with a sensitivity of about 105 cysts using a radiolabelled probe selected from a cloned Giardia DNA library.5 There was no cross-reaction with the closely related Trichomonas vaginalis or with Entamoeba histolytica (figure), or with Crithidia fasciculata or Leishmania donovani. Though the probe cross-hybridised with
05 µg of T cruzi DNA, this would not be a problem in practice, and at 50 µg the amount of mammalian DNA needed to give a signal was orders of magnitude greater than the amount of Giardia DNA. The microscopic detection of G lamblia cysts in faecal smears may be only about 50% successful,6 and cysts present in water may be missed at concentrations below 4000/1.7 Thus, DNA probes may offer advantages over traditional methods of detecting Giardia. With the introduction of non-isotopic labels we would advocate their wider use as diagnostic tools. Division of Communicable Diseases, St George’s Hospital Medical School, London SW17 ORE, UK
Department of Medical Protozoology, London School of Hygiene and Tropical Department of Pure & Applied Biology, Imperial College of Science, Technology & Medicine, London
D. J. M. LEWIS Medicine
E. L. GREEN
F. ASHALL
1. McManus DP, Simpson AJG. Identification of Echinococcus organisms using cloned DNA markers. Mol Biochem Parasitol 1985; 17: 171-78. 2. Greig S, Ashall F. Detection of South American trypanosomes in insects using total parasite DNA probes. Parasitol Today 1987; 3: 375-76. 3. Ashall F, Yip-Chuck DAM, Luquetti AA, Miles MA. Radiolabelled total parasite DNA probe specifically detects Trypanosoma cruzi in mammalian blood. J Clin Microbiol 1988; 26: 576-78. 4. Flisser A, Reid A, Garcia-Zepeda A, McManus DP. Specific detection of Taenia saginata eggs by DNA hybridisation. Lancet 1988; ii. 1429-30. 5. Butcher PD, Farthing MJG. DNA probes for the faecal diagnosis of Giardia lamblia infections in man. Biochem Soc Trans 1989; 17: 363-64. 6. Kamath KR, Murugasu R. A comparative study of four methods for detecting Giardia lamblia in children with diarrhoeal disease and malabsorption. Gastroenterology 1974; 66: 16-21. 7. Feachem RG, Bradley DJ, Garelick H, Mara DD. In: Sanitation & disease: health aspects of excreta and waste water management. London: John Wiley & Sons, 1983: 349-56.
Normal
development after exposure to mifepristone in early pregnancy
Results of hybridisation with the total genomic Giardia DNA
probe. 1 =Glamblia DNA 1.5 J.!g (A), 05 g (B), 0.1 J.!g (C), 10 ng (D), 1 ng (E), 2=vaginalis DNA 5 pg (A), 0 5 µg (B), 50 ng (C); 3=Ehistolytica trophozoites 6 x 105 (A), 6 x 10’ (B); 4=T cruzi DNA 5 µg (A), 0,5 µg (B), 50 ng (C), 5=rat DNA 5 wg (A), 0-5 pg (B), 50 ng (C); 6=G lamblia trophozoites 10. (A), 5x 105 (B), 105 (C), 10’ (D), 103 (E), 500 (F); 7 = G lamblia cysts 105 (A), 6x10" (B), 310" (C), 10"(D), 6x 103 (E)
SIR,-Mifepristone (RU38486), due to its ability to block the action of progesterone in the pregnant uterus, offers a novel approach to drug-induced termination of pregnancy.’ Given alone to terminate early pregnancy mifepristone is effective in about 80% of cases. The subsequent administration of subtherapeutic doses of a prostaglandin analogue increases efficacy to 95%. It can also be given alone as a cervical ripening agent before transcervical operative procedures. Mifepristone has teratogenic effects in the rabbit but not in other speciesand it may have been associated with a malformed fetus aborted in the second trimester after the mother took the drug to terminate an early pregnancy.3 We wish to report the outcome of the pregnancies of three women who decided to continue to term, despite having taken mifepristone during participation in one of two clinical trials. Cases 1 and 2 were recruited into a study of mifepristone and gemeprost in the termination of early pregnancy. Case 3 took part in a double-blind, placebo-controlled trial of the cervical-priming properties of mifepristone before surgical termination of pregnancy. All patients gave written informed consent. Case 1. A 21-year-old primigravida was given mifepristone at 8 weeks’ gestation. Before prostaglandin administration, the patient indicated that she did not wish to proceed with the termination. Throughout the pregnancy, ultrasound scans showed satisfactory fetal growth. The patient reported two episodes of light brown vaginal discharge. The pregnancy progressed satisfactorily, and the patient went into spontaneous labour at 40 weeks’ gestation and delivered a healthy boy weighing 4150 g. Postnatal examination of the infant revealed no abnormality and he continues to develop normally 15 months later. Case 2. A 31-year-old (para 2 + 0) was given mifepristone at 8 weeks’ gestation. Before administration of the prostaglandin she indicated that she did not wish to proceed with the termination. 2 days later the patient was admitted with vaginal bleeding and mild pelvic cramps. Bleeding settled after 3 days in hospital and ultrasound scan confirmed a viable pregnancy. Subsequent antental progress was uncomplicated. The patient was admitted in labour at 39 weeks’ gestation and delivered a healthy male infant weighing
![[Giardia lamblia or Giardia lambli?].](/assets/img/hold.png)
