Hattie E. Alexander

Treatment of type-specific hemophilus influenzae infections in infancy and childhood

Summary The clinical features of the following infections with type B H.influenzae are presented: meningitis, respiratory infection causing obstruction of the respiratory tract, and pneumonia complicated by empyema in infants under 1 year of age. A therapeutic program is outlined for each of these groups. The biology of H. influenzae is discussed for purposes of clarifying the principles which have been applied in treatment. Laboratory tests useful in the therapeutic program are presented. The results of the treatment advised in meningitis are analyzed from several points of view. Of the fifty patients reported, thirty-seven, or 74 per cent, recovered. The treatment of ten patients with obstructive infections of the respiratory tract is discussed.

Quantitative studies on the infectivity of ribonucleic acid from partially purified and highly purified poliovirus preparations

Abstract The method of preparation of infectious ribonucleic acid (RNA) from poliovirus and the factors that influence its degree of infectivity for cells in monolayers have been examined in some detail in an attempt to discover why the efficiency of RNA infection was so low. It was found that isolated RNA was quite stable to both phenol and ether extraction, suggesting that there is little degradation of RNA from the procedures used. The isolation of highly infectious RNA preparations from very pure virus demonstrates that its source can be the intact virus particle. A number of factors were found that influenced the efficiency of RNA in infecting human cells in monolayers. The exposure of host cells to hypertonic salt solutions prior to seeding increased their susceptibility to RNA infection to a significant degree. The salt concentration and pH of the RNA inoculum showed the most striking effect. Under optimum conditions the number of plaques formed was found to be directly proportional to the input of RNA. The maximum plaque-forming efficiency of RNA preparations was 1.0% of the virus from which they were removed.

Validity of etiological diagnosis of pneumonia in children by rapid typing from nasopharyngeal mucus

Summary Nasopharyngeal mucus obtained through the nares and emulsified in0.2 c.c. of pneumococcus broth serves as excellent material for direct typing of pneumococci and H. influenzae by the Neufeld method. Cultures of this material show a striking predominance of the etiological agent. Three hundred and seventy-seven patients have been studied by this method: 317 cases, or 84 per cent, were diagnosed in less than twelve hours; 258 cases, or 68 per cent, diagnosed in thirty minutes; 60 cases showed negative results in twelve hours, but within a twenty-four-hour period, 55 per cent of these showed a predominance of Streptococcus hemolyticus by culture. Establishment of the etiological agent by the method outlined hasbeen satisfactory even when carried out by a number of observers and in most instances upon one specimen of nasopharyngeal mucus. Twenty-seven of the ninety-eight lung punctures yielded positive cultures. The organisms isolated are listed. There were no instances of empyema following lung puncture. Two patients developed pneumothorax subsequent to lung puncture, but both recovered uneventfully within a ten-day period. The objective evidence available in forty-five cases showed confirmation in forty-three, indicating 95 per cent agreement. The experience with lung puncture indicates that it is a relativelyharmless procedure.

Type “B” Anti-influenzal Rabbit Serum for Therapeutic Purposes

The treatment of influenzal meningitis in children with sera from horses immunized with H. influenza has, in general, been unsuccessful. The investigations of Goodner and Horsfall 1 , 2 , 3 demonstrated the superiority of rabbit over horse antipneumococcal serum. The causes of this superiority were believed to be, first, the smaller size of the anticarbohydrate molecule of rabbit serum, endowing it with greater powers of penetration of the tissues, and secondly, the absence of the prozonal phenomenon 4 or inhibitory mechanism in rabbit serum, when amounts greater than a critical quantity were used. Rabbit anti-influenzal serum, because of its greater penetrating powers, and some evidence that it crossed the blood-brain barrier, seemed to offer theoretical advantages over horse serum, assuming that the important antibody of the anti-influenzal serum is the anticarbohydrate as in rabbit antipneumococcal serum. Pittman 5 has brought forth much evidence that there is a parallelism between the antigenic structure of pneumococci and H. influenza. She demonstrated a capsule on all smooth strains by Muirs capsule-stain and showed that such strains had the power to form soluble type-specific substances. All strains were found to be bile-soluble and therefore apparently produced enzymes as does the pneumococcus. Because of this similarity, the knowledge derived from the study of pneumococci has been applied in the selection of a suitable antigen for the production of rabbit antibodies for H. influenza. The strains used were those isolated from cases of influenzal meningitis. All the meningeal strains from children thus far reported have been Type B. Typing was performed by the capsular-swelling method with antisera obtained from Pittman. A large loopful (3-4 mm) of Type B antiserum is placed upon a coverslip. To it is added a 1 mm loopful of dilute young culture or biological fluid containing the suspected H. influenza organisms.

Production of poliomyelitis virus with combined antigenic characteristics of type I and type II

Abstract Evidence is presented indicating that a “combined” poliomyelitis particle which can be neutralized by type I as well as by type II antiserum has been produced in tissue cultures after simultaneous infection of monkey kidney cells with type I and type II poliomyelitis viruses. Serial passage of the “combined particles” suggests that they are capable of reproducing as such but are not stable.

Infectivity of Ribonucleic Acid (RNA) from Type I Poliovirus in Embryonated Egg.

Summary 1) Phenol extraction of Type I poliovirus yielded a plaque-forming agent which is presumably ribonucleic acid (RNA) because it is inactivated by RNAase. Such ribonucleic acid derived from Type I (Mahoney) poliovirus induces formation of plaque-forming agent in the embryonated egg, inoculated by amniotic route. The plaqueforming agent recovered from embryo tissue, sacs, and fluids has been identified as type specific, Type I poliovirus. Highest yields occurred at 6 to 8 hours after inoculation of eggs with polio-RNA. 2) Polio-RNA exposed to RNAase immediately before inoculation into embryonated eggs failed to incite formation of plaque-forming agent. This establishes that it is RNA itself, and not some undetectable trace of intact virus, which incites production of whole virus in the egg. 3) Propagation of virus recovered from eggs by further passage in embryonated egg have not succeeded. 4) This demonstration of production of Type I poliovirus from nucleic acid in the embryonated egg, without subsequent multiplication, provides a system for biochemical study of genesis of whole virus from its nucleic acid. An advantage of the system is that the inciting agent (infectious RNA) can be differentiated completely, by its susceptibility to the action of RNAase, from whole virus progeny.

The present status of treatment for influenzal meningitis

Abstract A larger experience and more time for long term evaluation of physical and mental development are needed before final statements can be made concerning the results of treatment of H. influenzae infections. Certain facts are evident, however. In patients in whom the infection is mild or moderately severe, according to the criteria previously described, either streptomycin alone or sulfadiazine in conjunction with specific rabbit antiserum can be expected to cure 100 per cent of them. A small fraction can be cured by sulfadiazine alone. When manifestions of severe infection are present in patients in whom it is evident from the history that the onset of the meningitis can be dated within a few days, the results suggest that a choice may be made between two therapeutic programs, the combined action of sulfadiazine with either streptomycin or type b H. influenzae antiserum. Experience with the latter regimen is so extensive that one can predict complete recovery in virtually 100 per cent of these patients. The use of the former program, simultaneous use of sulfadiazine and streptomycin, is still too limited to recommend it with assurance in this group, though its success is anticipated. Patients whose infections have progressed to the severe state despite the use of sulfonamides cannot be considered suitable cases for treatment with sulfadiazine in conjunction with streptomycin; the presence of sulfadiazine resistant H. influenzae in significant numbers may prevent these agents from eliminating the meningeal infection. Moreover a longer period of study on the toxic effect of streptomycin on the central nervous system is necessary before it can be recommended as the treatment of choice for this group even if it proves to be equally effective. Those patients with severe meningitis and a history which suggests that uncontrolled meningitis has been present for a week or more or who show signs of chronic meningitis with or without manifestations of localized cerebral injury should receive sulfadiazine, streptomycin and specific rabbit antiserum simultaneously. The latter program can be expected to reduce the risk of failure to a minimum since it combines the action of three antibacterial agents which exert their destructive influences through three different mechanisms. Members of the population resistant to one can be attacked by another or both the others. The difficulty in determining the time of onset of meningitis in young infants under seven months of age, because of failure of these infants to exhibit signs of meningeal infection until several days after onset, together with the still high mortality rate in this group, have led us to recommend the use of sulfadiazine, streptomycin and specific rabbit serum initially for these infants. It is in this severest group that the addition of streptomycin to sulfadiazine and specific antiserum can be expected to reduce the 20 per cent mortality which resulted from the combined use of the last two agents. The use of streptomycin alone or in conjunction with sulfadiazine is justifiable only when laboratory facilities permit evaluation of severity of infection and progress of recovery. The need for bacteriologic methods which can detect small numbers of viable organisms and evaluate sensitivity of any culture grown from the spinal fluid, is of paramount importance.

Purification of the desoxypentose nucleic acid of Hemophilus influenzae having transforming activity

Abstract The purification of DNA preparations of Hemophilus influenzae , types b and c, endowed with transforming activity is described. The mild procedure makes use of electrophoretic separation of the DNA from contaminating PNA and polysaccharides. The final DNA preparations, free of detectable amounts of these contaminants and of proteins, were active in transformation in concentrations of less than 0.0004 μg. and 0.01 μg. DNA/ml. for types b and c, respectively.

Experimental investigations as a basis for treatment of type B hemophilus influenzae meningitis in infants and children

Summary Experimental results demonstrate that there is a close correlation between the mouse protective capacity of sulfonamides and their degree of inhibition in the test tube against type b H. influenzae. Sulfadiazine is vastly superior to sulfanilamide and definitely more effective than sulfapyridine. A simple in vitro test is described for estimating the susceptibility of a given strain to sulfadiazine within forty-eight hours of isolation. Sulfadiazine and type-specific rabbit antibody, when used singly,have been shown to possess approximately equal protective value against lethal type b H. influenzae mouse infections. However, regardless of the dose administered they fail to protect regularly when the inoculum exceeds 10,000 minimal lethal doses. Mice treated with both agents together quite regularly survive following the injection of 1,000,000 minimal lethal doses. It seems quite clear that in the treatment of patients with influenzal meningitis the risk of failure is reduced to a minimum by the use of both serum and sulfadiazine. However, if the infection is judged mild by clinical and laboratory criteria, initial treatment with chemotherapy alone is justified, provided the recommended precautions and guides are used and provided the strain shows average susceptibility to sulfadiazine.

Streptomycin in pediatrics

Summary Analysis of the published data shows lack of agreement between in vitrosensitivities and the clinical response to streptomycin of a number of human infections. Employment of an in vitro method which applied certain principles—use of a large inoculum (3 to 1,700 million organisms), an optimal medium (Levinthal broth or agar), and incubation period of 48 hours—yields sensitivities which are in better agreement with the available therapeutic results. Such statements must be accepted as only tentative until adequate clinical experience makes a final appraisal possible. The method offers a basis for comparison of all gram-negative rods studied. The test has provided a reliable index of the therapeutic efficacy of streptomycin in influenzal meningitis in which clinical trial does permit certain conclusions. The narrow range of sensitivity of all strains of H. influenzae studied prior to exposure to streptomycin is in agreement with the prompt recovery of patients who have meningitis of mild or average severity. On the other hand, in the severe cases therapeutic results with streptomycin alone have been disappointing; in two of the five unsuccessful cases the failures were due to emergence of resistance of the strains. It is already clear that the therapeutic efficacy of streptomycin againstmost varieties of susceptible severe infections will be significantly influenced by emergence of resistance of the organisms.