THE LANCET, OCTOBER

25,1975

rnorenineemic patients with renovascular hypertension

may be explained by hypotheses provid ing for renin-angioten sin dependence of hypertension without absolute elevation of peripheral renin levels. I I 11 2 patients with high-renin essential hypertension also showed a depressor response in the saralasin test. Thus, were the test used as a preliminary screening procedure for reno vascular hypertension, 2 of 13 saralasin responses in this series would have been interpreted as falsely positive--i.e., the pat ients would have been subjected to needless further investigations before a correct diagno sis became apparent. These responses constitute direct in-vivo evidence that the renin -angiotensin system does maintain the elevated pressur es in high-renin pa, tients with chronic ben ign hypertension and no ren al or renovascular lesions. This finding would app ear to refute MacGregor's recent observation that saralasin does not lower blood-pressure in patients with essenti al hypertension even when they are sodium-depleted. 13 Whet her or not saralasin response will occur in all 2-3 4% of essentia l hypert ensive pa tient s classified by others as a high-renin subgroup!' rema ins a key su bject for future investigations. Another potential source of misleading test results might be a patient with highrenin essential hypertension and an incidental renalarte ry stenosis. T he importance of concomitan t sodium depletion, achieved in this study by giving the patient furo semide the evening before th e test, deserves further emphasis. Donker and Leenan recently reported 2 patients with renovas cular hypertension (confirmed by successful operati on) who preoperatively failed to respond to saralasin infusion.!" Neither patient was salt-depleted. Before operation, 1 pat ient was reinfused with sara lasin in a salt-depleted state, and he then showed a depr essor response. Prior studies in our patients without salt depletion support this observation: instead of 13 saralasin responders, only 7, each with a rather blunted response, were noted. These observations pro vide strong support for angiotens in-sodium interaction in reno vascul ar hypertension. IS 16 The present data also suggest that the pharmacological half-life of saralasin is much longer than previously stated. In 2 patients showing depre ssor responses to a saralasin bolus, Pettinger et al. found the half-life of return of blood-pressure to pre-bolus levels to be 4·1--4·3 minute s.!? In the present group of 13 responders, very little return of blood-pressure is noted for as long as 30 minutes of observat ion after th e bolus. However, Pett inger 's pat ient s were not salt-depleted and were ingesting multiple antihypertensive medicat ions at the time of the test; they also received a smaller dose of saralasin than the present patients. Duration of depressor response to the saralasin bolus needs further clarification before widespread outpatient usage can be recommended. The saralasin bolus test has man y characteristics of an ideal screening procedure for ren in-mediated hypertension. When performed as described herein, the test is rapid , safe, simple, inexpensive, easy to interp ret, and devoid of falsely negative responses. Moreover, it gives direct in-vivo evidence of the role of the renin-angiotensin system in susta ining a hyperten sive state. Such information, heretofore available only by inference, is critical in the diagnosis of renovascular hypertension and may also be important in the prognosi s'" and choice of

787 therapy'? of the larger population of pat ients with essential hypertension. We gratefully acknowledge the assistance of Cheryl Gross and Janet Rotenberg, registered nurse s, who performed th e sara lasin studies, and of Graciel a Vegagomez who performed the renin assays. This work was supported in part by the University Medical Resear ch Foundation and U.S.P.B .S. RR00865. Saral asin was gener ously pr ovided by Dr Robert Keenan and Dr Joseph Dinardo, Eaton Laboratories, Norwich, New York. Requ ests for repr ints should be addressed to M.H .M., Cedars-Si nai Medica l Center, 8720 Beverly Blvd., Los Angeles, Ca lifornia 90048, U.S.A.

REFERENCES I. Rru nner, H. R., Gav ras, H., Larag h, 1. n., Keenan. R. Lancet , 1973. ij , 1045. 2. Streeten, D. H. P., Anderson, G. H., Freiberg, J. M., Dala kos, T . G. N ew Engl.]. Med . 1975,292, 657. 3. Pals, D. T ., Masucci, F. D., Denning, G. S., Sipos, F., Fessler, D. C. Circulation R es. 1971,29,673 . 4. Pals, D. T ., Masucci, F. D. Eur, ] . Pharmac. 1973,23, 115. 5. Stre eten, D. H. P., Freiberg, J. M" Anderson, G. H., Dalakos, T . G. 'Circulation R es. 1975, 36/ 37, suppl. I , p. 125. 6. Hoch berg, H . M., Salomon, 11. Curro th er, Res. 1971, 13, 129. 7. Haber , E., Koerner, T ., Page, L. B., Kliman, B., Pumode , A.J. d in. Endocr. 1969, 29, 1349. 8. Maxwell, M. H., Marks, L. S., Varady, P. D., Lupu , A. N.. Kaufma n, j . j . 7. Lab. d in, M ed. (in th e press), 9. Maxwell, M. H., Bleiffer , Fra nklin, S. S.. Varad y, P. D. J. Am . med. A ss. 1972, 220, 1195. 10. Mark s, L. S., Maxwell, M . H. Urol. Clin. N. Am . 1975, 2, 31t. 11. Dunn , M. j ., Ta nnen, R. L. Kidney Int. 1974, S, 317. 12. Blaqu ier, P., Bohr, D. F., Hoobler, S. W. Am .]. Phy siol. 1960. 198, 1148. 13. MacG regor, G. A., Markandu , N. D:, Dawes, P. M. Proceedings of the Sixt h Interna tional Cong ress of Nephrology, Florence, Italy, 1975, p. 519. 14. Denk er, A. J. M., Leenen , F. H. H. Lancet, 1974, ii, 1535. 15. Brunn er, H. R., Kirshrnan , j . D., Sealey, J. E., Lar agh, j . H. Science, 197 1. 174,1344. 16. Gavras, II ., Bru nner, H. R., Vaugha n, E. D., Lara gh, J. H. ibid. 1973,180, 1369. 17. Petti nger, \'('. A., Keeton, K., Tanaka, K. Clin . Pharmac. Th er. 1975. 17, 146. J 8. Brun ner, H. R., Larag h, J. u., Baer, L.. Newton, M. A., Goodwin, F. T ., Krakoff, L. R., Bard, R. H., Buhler, F. R. Ne w Engl .]. M ed. 1972, 286, 441. 19. Buhler, F. R., La ragh, j. H., Baer, L., Vaugha n, E. D., Bru nner, H. R. ibid. 1972,287,1 209.

AUTOMATION OF SALMONELLA TYPHI PHAGE TYPING

J. J.

FARMER,

III JANET

FRANCES SIKES

W.

HICKMAN

V.

Bacteriophage-Bacteriocin Laboratory, Center for Disease Control, Atlanta, Georgia 30333, U.S.A.

Two steps in the procedure for bacteriophage typing of Salmonella typhi have been automated. The culture inoculum was applied by flooding the surface of phage agar in a 150 x 20 mm petri dish and removing the excess liquid with a safety pipettor. This step replaced the older method of manually preparing up to 100 individual areas for inoculation. The number of bacteria per unit area was the same with both methods, so the automated method involved no change in the technique. The second automated step involved use of a bacteriophage applicator which simultaneously dispensed 59 uniform drops of bacteriophages onto the inoculated plate. The automated procedure has reduced personnel time by about 90 % with no loss in sensitivity or accuracy. S

ummary

Introduction BACTERIOPHAGE

typing remains the method of choice

788

THE LANCET, OCTOBER

25, 1975

0·01 ml loop and gently spread in circles with diameters of 1-1'5 em, The phages are applied to the inoculated area with the same loop, which is sterilised between each inoculation. Some time is saved by combining the phages into pools,t,. but many strains must be tested individually against all phages. 1. S A plate prepared by hand is shown in fig. 1; 50 manual operations were required to prepare it (25 to inoculate the bacteria and 25 to inoculate the bacteriophages).

Fig. I-Results from plate prepared by manual method (phage type T).

for subdividing strains of Salmonella typhi into epidemiologically significant subgroups. Over fifty countries 1, S use standardised reagents distributed by the International Reference Laboratory for Enteric Phage-typing at Colindale.! It has been 27 years since Craigie and Felix made suggestions for the standardisation of S. typhi bacteriophage typing. 3 During this time impressive technological advances have been made in many areas of science, yet bacteriophage typing of S. typhi is still done as described in 1938.' Then only 11 bacteriophages were used, so manual application was not a problem; however, today there are about 100 different bacteriophages in the typing schema. We have found that typing can be automated with no loss of accuracy or sensitivity.

Materials and Methods Manual Method This method 1,3-. is essentially the same as that described by Craigie and Yen in 1938.' The strain is grown in nutrient broth for 2-4 h and then spotted onto "phage agar" (Bacto nutrient broth 20 g, sodium chloride 8·5 g, agar agar 20 g and distilled water 1000 ml) with a standard

" -

Automated Method The procedure described above is used to grow the culture. A tube (4'5 ml) of the culture at the correct turbidity is then used to flood a dry phage agar plate (150 x 20 mm plastic petri dish). The plate is rotated carefully to avoid spillage until the inoculum is spread evenly over the surface, and the 'excess fluid is removed with a safety pipettor. Approximately 1 ml of fluid is absorbed into the agar. The plate is then placed vertically against a support for about 1 min. During this time, two other plates are prepared. Any remaining fluid is then removed from the bottom of the plate with two cotton swabs. All pipettes, tubes, and swabs are discarded into disinfectant, and care is taken to avoid spills or aerosols. The plates are dry within a few minutes and the phages are applied. The device used to deliver the bacteriophages is shown in fig. 2. The aluminium stand contains two movable parts-a petri dish support (S) which can be raised and lowered and a pressure plate (P) which is lowered by a screw mechanism. As the pressure plate is lowered, it exerts pressure on the syringes and a single drop of bacteriophage is delivered from each one. The 3 ml autoclavable syringes (Sherwood Medical Industries, Deland, Florida 32720) are fitted with 38 mm (1·5 in), 25-gauge hypodermic needle (Beckton, Dickinson, Rutherford, New Jersey 07070). A short piece of ' Teflon' tubing with an inside diameter of 0·38 mm (0'015 in) (Bel-Art Products, Pequannock, New Jersey) is placed over each needle to ensure uniform drops. After the syringes have been autoclaved and filled with phage they are placed into a removable aluminium plate (A). After one drop of each phage has

_ .,...

Fig. 2-Bacteriophage applicator filled with 59 syringes for plate I; an individual syringe fitted with a needle and covered with teflon tubing; the syringes for plate 2.

THE LANCET, OCTOBER

789

25,1975

been applied in a single operation, the aluminium plate is removed to the refrigerator. Thus we can use one phage applicator for other phage-typing systems. With the phage applicator, 59 uniform drops of about 0·01 mI are delivered in a single step. The pattern of the phages applied to plates 1 and 2 is shown in fig. 3. In plate 1 SI/S8 are eight unadapted Vi phages (these lyse only stains of S . typhi which contain the Vi antigen); A x 20 is phage A at 20 times its routine test dilution. In plate 2, 01 is the bacteriophage described by Felix and Callow 6 and used by Cherry et a!.7 for identification of Salmonella. PI is a pool of the last 10 bacteriophages (DI2, Ell, F6, G4, G5, 51, 52, 53, 54, 55) added to the typing schema. All the other designations are those normally given for the typing phages.

Results We have used the automated method in our laboratory for 2 years and feel that it has many advantages over the manual method and no disadvantages. Uniformity of Lawn Standardised phage-typing demands the preparation of an even lawn of the test strain. Plate flooding and inoculation by standard loops are different methods, but

e

51

,

r

n

fI

5

•

II "

4

~

.l

II .It

U

.,

M

n

PLATE 1

PLATE 2

Fig. 3-Patternof phages applied to each plate.

TIME REQUIREMENTS FOR TYPING FOUR DIFFERENT PHAGE TYPES BY MANUAL AND AUTOMATED METHODS

Time No. of set-up steps

Phage typ e

Method

A

Automated Manual

200

Automated Manual

200

Automated Manual

73

Automated Manual

78

Degraded Vi EI E9

4 4 4 4

requi red for set-up

Time (h) for final report after culture has grown

5 50

8 32

5 50

8 32

(min)

5 18 5 19

8 88 32

- Presumptive report.

the result is the same-a uniform lawn with the same number of bacteria per unit area. A culture of density 10 8 bacteria{ml inoculated by 0·01 ml standard loop and spread in a circle of diameter 1·34 em (mean value visually measured from photographs) will yield a density of 7·1 x 10' bacteria/em", If the plate is flooded it takes up 1 ml of culture fluid, and the resulting density for a plate 14 em in diameter will be 6·5 x lOS bacteria/ems. Thus the two procedures agree within 10%, which is excellent agreement considering all the other possible variables. The other requirement in phage typing is that a standard number of phage particles be applied for each test. Inoculation with standard loops or with standard syringes clearly accompli shes the same goal, and the phages are stable in the syringes for at least 6 months. Time Required The automated method saves time by shortening the processing procedure by 75-90%, and by virtually eliminating phage pools. Often the final report is ready 24 h earlier than with the manual method (table). Quality Control Quality control is facilitated with the automated method. With each typing run we test each phage against Vi phage type A which is lysed by each one of the phages. Thus in each typing run we can tell if any of the phage preparations has significantly dropped in titre. The automated procedure also makes it very easy to test new lots of nutrient broth and agar-agar to determine if they are satisfactory for the typing procedure. This is an important consideration. 6 Lately several batches of agar-agar have given poor results. We tested 12 different lots of agar-agar from six different suppliers and found that 6 of 12 were unsatisfactory because they inhibited the lysis of as many as 50 % of the phages. Safety

Fig. 4-Typing patterns of phage types A and HI prepared by automated method.

Upper pair of plates shows pattern for phage type A, and lower pair is for type E 1.

Safety officers at the Center for Disease Control ruled (copy available on request) that it was not necessary to use a biological safety cabinet for our automated procedure. Aerosol formation in the automated procedure is no greater than in the manual one . Settling plates set out during the procedure have never been positive for S. typhi, Only normal safety rules are enforced (no food or drinks, no smoking, no mouth pipetting, replacement of gas burners

THE LANCET, OCTOBER 25,1975

790 with safety incinerators, current typhoid immunisations, and thorough hand-washing after handling S. typhi cultures). The risks of laboratory-acquired typhoid fever are small through the aerosol route, and adherence to good laboratory technique can minimise risks of oral ingestion. Other Ramifications By choosing the phages representing more common phage types and using pools, it would be possible to do a screening on one plate instead of the normal two. This approach has been tried in our laboratory with good success. For laboratories with many cultures to process it may be very desirable. The automated typing procedure can accommodate new typing phages because there are places for 20 additional syringes on the second plate. New phages that are added to the typing procedure can be added either individually or as pools. Supplemental typing phages to subdivide the more common phage types could also be added. Typing phages for subdividing phage type A and "untypable Vi" strains have been described." Discussion The main disadvantage of S. typhi phage typing is the time the procedure takes. Edwards and Wilson 5 used screw-cap bottles fitted with dropping pipettes to disperse bacteriophage suspensions. This method replaced the older one of using wire loops which had to be sterilised after each use. Nicolle 9 described a method for applying the bacterial inoculum uniformly to 100 x 100 mm agar plates by flooding and removing excess fluid. Our method is a refinement of these attempts to automate the procedure. The results are identical to those 'obtained with the manual procedure. This is not surprising because both methods deposit the same number of bacterial cells per unit area and deliver the same number of bacteriophage particles onto the lawn. The rest of the procedure remains unchanged. The automated method has been used to type over 1000 cultures and we have found no disadvantages. The main advantage is the 75-90% reduction in the time required for processing samples. The bacteriophage applicator is an invaluable asset to our laboratory, and we use it for phage typing of S. typhi, S. typhimurium, Serratia marcescens, Shigella, Escherichia coli, and Proteus mirabilis as well as for bacteriocin typing.w The phage applicator is available commercially from Johnny Brown Machine Shop, P.O. Box 239, Route 3, Tuscaloosa, Alabama 35401, U.S.A., or a set of machine drawings for its construction can be obtained from J. J. F. Requests for reprints should be addressed to J. J. F.

PRENATAL DIAGNOSIS OF HOMOZYGOUS ~- THALASSlEMIA YUET WAI KAN MITCHELL S. GOLBUS RICHARD TRECARTlN Departments of Medicine, Obstetrics and Gynecology, and Clinical Pathology and Laboratory Medicine, University of California, San Francisco, California, U.S.A.

MARIO FURBETTA ANTONIO CAO Padiatric Clinic, University of Cagliari, Sardinia, Italy

In two pregnancies at risk for homozygous ~+ and ~o thalassemia, fetal blood mixed with maternal blood was obtained by placental aspiration and was purified to over 90% purity by differential agglutination with anti-i antibodies. Study of globin-chain synthesis showed absence of ~-globin chain in both fetuses. The diagnosis of homozygous ~-thalassremia was made and was confirmed after the pregnancies were terminated. Thus, the defect in ~-glo­ bin-chain synthesis in homozygous ~-thalassremia is expressed in utero, and prenatal diagnosis is possible. Summary

Introduction MEASUREMENT of globin-chain synthesis in fetal blood is being utilised as a method for prenatal diagnosis of sickle-cell ansernia and thalassemia. I 2 Several fetuses aborted for psychosocial reasons have been identified as having sickle-cell trait, 1-4" and the determination of the homozygous state, in which only ~-s and no ~-A chain would be present, should not pose any difficulty in biochemical analysis. In ~-thalassremia the diagnosis depends on the demonstration of a quantitative reduction in ~-globin-chain synthesis. An unanswered question was whether the ~-giobin-chain synthesis defect in homozygous ~-tha­ lasssemia was expressed in utero. In studying mixed fetal and maternal blood from fetuses of parents with ~-tha­ lasssernia trait aborted during the first trimester, Chang and his coworkers found several samples with reduced ~-globin-chain synthesis.' However, confirmatio~ of ~-thalassremia was not possible in these earl.y abo~ttons. We describe here two cases where prenatal diagnosis was attempted in pregnancies at risk for ~+ and ~o thalasssemia. Study of fetal blood, obtained in utero in one case at the 21st week and in the other in the 18th week of gestation, showed absence of ~-globin-chain synthesis in both fetuses. The diagnosis of homozygous ~-thalassre­ mia was made and was confirmed after the pregnancies were terminated.

REFERENCES

Materials and Methods

1. Anderson, E. S., Williams, R. E. O. J. clin. Path. 1956, 9, 94. 2. International Committee for Enteric Phage-Typing, J. Hyg., Camb. 1973,71,59. 3. Craigie, J., Felix, A. Lancet, 1947, i, 823. , 4. Craigie, J., Yen, C. H. Can.]. publ. Hlth, 1938,29,448. 5. Edwards, P. R., Ewing, W. H. Identification of Enterobacteriacee. Minneapolis, 1962. .. 6. Felix, A., Callow, B. R. Br. med.J. 1943,11,127. 7. Cherry, W. R, Edwards, P. R, Hogan, R R J. Lab. clin. Med. 1954,44,51. 8. Nicolle, P., Paylatou, M., Divemeau, G. Ann. Inst, Pasteur. 1954, 87,493. 9. Nicolle, P. J. Egypt. publ. Hltb Ass. 1970, 45, 119. 10. Farmer, J. J" III. Appl. Microbiol. 1972,23,218.

The first patient has one child with homozygous p>thalas~z­ mia (p/iX=O·I), and the second patient has two children with homozygous pO-thalasszmia. After genetic counselling both families requested prenatal diagnosis. Fetal blood samphng was attempted at the 21st and the 18th week of gestation respectively, by placental aspiration using a ~O-gau~e dispos~ble spinal needle. Small samples of blood mixed With ammonc fluid were withdrawn and analysed on the Coulter counter particle-size analyser." 7 In the first patient tWO sa~ples containing 30 and 15% fetal blood, and in the second patient one sample containing 25% fetal blood, were obtained. These samples