■■■

王

第22巻第3号平成6年9月

内
原

容

著

HTLV−1感染者において脳MRI検査により確認された潜在性の広汎な白質変化（英文）・山内 慎介，伊地知信二，新名 清成，法化図陽一
丸山 征郎，新村健，納光弘……………… 117−120
ダッカ（セネガル）における新生児破傷風の予後に与える疫学的因子（英文）・・PapaSalif

Sow，Bemard

MarcelAwa

Faye，Salif

Badiane

Mame
and

Awa

MarineDiop，

Coll−Seck………・………・……一

121−124

インドネシアスラバヤ地区における小児下痢便の解析（英文）・・仲宗根昇，岩永正明，Eddy

Bagus

Wasito，

PitonoNyomanGdeRanuh

125−127

Sithivong，岩永正明

129−131

SoepartoandI．Gusti

日本とラオスで分離された黄色ブドウ球菌の比較研究（英文）・・比嘉直美，Noikaseumsy

ニューモシスチス・カリニ肺炎の病原性発現におけるニューモシスチス・カリニ

1

管状突起の役割（英文）

・・塩田
有園

恒三，嶋田

義治，栗本

浩，及川

直樹…・……………・……………・・…・…・………

弘
133−137

会報・記録
1994年度会員名簿…・…・…………・……一………・

一139−160

投稿規定一………………・…・・……………・………

・・161−162

日本熱帯医学会雑誌編集委員名簿………………

−163−1641994年度役員名簿……・…・………・………………

・・165−166「第6回熱帯医学と医療を語る会」お知らせ…・−167

第24回日本医学会総会1995（名古屋）広報NQ3・

−168

1■

117

Jpn. J. Tro p. Med. Hyg., Vol. 22, No. 3 (1994) pp. 117 120

OCCULT DIFFUSE INVOLVEMENT OF

SUPRATENTORIAL WHITE MATTERDETECTED BY MAGNETIC RESONANCEIMAGING IN HTLV‑ I CARRIERSSHlNSUKE YAMAUCHI', SHlNJI IJICHI*, KIYOSHIGE NIlNA',

YOICHI HOKEZU*, IKURO MARUYAMA*, TATSURU NIIMURA*
and MITSUHIRO OSAME*
Received April 27 1994/Accepted June 28 1994

Abstract: We describe three human T Iymphotropic virus type I (HTLV‑ I ) carriers, who are character‑ized by a dissociation between the clinical features including scarce cognitive impairments and peculiar
hyperintensities in the deep and subcortical white matter detected by T2‑weighted magnetic resonanceimaging (MRD scans. Unlike spotty MRI findings which are encountered in clinically asymptomatic elderly
individuals and are previously described in some HTLV‑ I carriers, the MRI changes in our patients werediffuse and extensive. Known central nervous system disorders accompanying a cerebral white matter
involvement were failed to be diagnosed. These findings support a possibility that extensive lesions in the

supratentorial white matter are associated with HTLV‑ I infection with minimal symptoms, and suggestthat MRI scans may detect occult diffuse inflammatory changes associated with the virus infection in thesepatients.

Keywords: Human T Iymphotropic virus type I (HTLV‑ I ) ; Cerebral white matter; T2‑weighted irnage;
High intensity.

INTRODUCTION
Sustained prevalence of human T Iymphotropic
virus type I (HTLV‑ I ) , a member of the oncoviridae
subfamily, is common in many parts of the tropics andin southern Japan (Osame and McArthur, 1992). The
virus causes two well defined diseases: adult T cellleukemia/lymphoma, which is a neoplastic disease char‑acterized by clonal expansion of HTLV‑ I ‑transformed
T cells, and HTLV‑ I ‑associated myelopathy/ tropicalspastic paraparesis (HAM/TSP) . The latter is a non‑fatal neurological syndrome characterized by predomi‑
nant involvement of the spinal cord and manifested bymatter of HAM/TSP patients (Izumo et al., 1989).
Cerebral white matter involvement detected by mag‑netic resonance imaging (MRD has been reported in an
HTLV‑ I infected symptomless individual (Mattson etal., 1987) and the incidence of cerebral MRI abnormal‑
ities is significantly higher in patients with HAM/TSP
than in the controls (Kira et al., 1988; Furukawa et al.,1989). These observations suggest that occult involve‑

ment of the cerebral white matter may be associatedwith HTLV‑ I infection. In previous cerebral MRI
studies on HTLV‑ I infected individuals, common
abnormal findings were multiple spotty high intensities

chronic spastic paraparesis. Although cognitive impair‑ment is included in less common neurological findings inin deep and subcortical areas on T2‑weighted images(Kira et al., 1988; Furukawa et al., 1989). Moreover,diffuse white matter involvement detected by MRI wasthis disease (Osame and McArthur, 1992), several ne‑

also described in connection with pyramidal tract sigus

cropsy studies have revealed that the critical pathologi‑

and cognitive dysfunctions in patients with HAM/TSP

cal findings including mononuclear infiltration or per‑ivascular cuffing, which is prominent in the thoracic(Natori, '1989; Valderrama et al., 1989; Uyama et al.,
1991; Konagaya and lida, 1991). Here we report threespinal cord, are commonly seen in the cerebral white

non‑demented HTLV‑ I carriers, whose cerebral MRI*Third Department of Internal Medicine, Faculty of Medicine, Kagoshima University, Kagoshima; 2Division of Internal Medicine,Kagoshima City Hospital, Kagoshima; and 'Kagoshima Red Cross Hospital, Kagoshima, Japan

118

c

A

B

Figure l. T2‑weighted axiai magnetic resonance images (spin echo relaxation time 2000, echo delay

100) of the patients demonstrate abnormal high intensity diffusely distributed in the
supratentarial white matter. A; patient 1. B; patient 2, and C; patient 3, respectively.

revealed diffuse and widely distributed white matterhigh intensity on T2‑weighted scans, and two of whomwere free from apparent features of pyramidal tract

Wechsier adult Intelligence Scale (WAIS). Routine

im pairment.

laboratory examinations including coagulation studies

CASE REPORTS

Routine neurological examinations disclosed no abnor‑
mality. Her intelligence quotient (IQ) was 102 on thewere within normal limits. The EEG record showednormal results. Cerebral CT scans revealed diffuse
hypodensities in the deep white matter, and MRI scans

Patient 1. A 74‑year old man presented with a 34
‑year history of slowly progressive gait disturbance.
Neurological examinations revealed spastic paraparesis

demonstrated high intensity signals diffusely distributedwith brisk tendon reflexes and extensor plantar

hypertension presented with a hand tremor. Neur‑

responses. Vibration sensation was decreased on bothlegs. The score of Mini‑Mental Examination was 26/30.
Routine laboratory examinations including coagulation

ological examinations revealed mild bradykinesia, mild
hyperreflexia without extensor plantar response, andorthostatic hypotension. Her IQ was 100 on the WAIS.
Routine laboratory examinations including coagulation
studies were within normal limits. Cerebral CT scansstudies were all within normal limits. Electroencephalo‑gram (EEG) showed a poor organization and modula‑tion. Myelography and myelo‑computed tomography(CT) scans demonstrated the cervical ossification ofin the white matter on T2‑weighted images (Figure IB) .
Patients 3. A 73‑year‑old woman with a history of

disclosed diffuse hypodensity in the deep white matter,and MRI scans revealed diffuse high intensity signals inposterior longitudinal ligament (OPLL) with canalstenosis. MRl scans revealed diffuse high intensity

the white matter on T2‑weighted images (Figure IO .

signals in the deep and subcortieal white matter on T2‑weighted images (Figure IA) .
Patients 2. A 86‑year old woman with a history ofantibody titers in serum (1: 512, 1: 2048, and 1: 65536,

respectively) , and in patient 3 the antibody was alsoatrial fibrillation and episodes of angina pectorispresented with a transient weakness of her right arm.

cle agglutination (PA) method (Fujirebio). The CSFS
obtained from patients I and 2 were negative for the

These 3 patients showed positive antiHTLV‑ ldetected in cerebrospinal fluid (CSF) (1: 2028) by parti‑

119

antibody (PA) . Western blot analysis confirmed thepresence of antibodies to HTLV‑ I antigens derived

MRI abnormalities are not related to apparent pyrami‑
dal tract signs at least in some cases. These observa‑

from MT‑2 cell line in sera both as lgG and lgM in thesethree patients (data not shown) . Oligo clonal lgG was
detected in CSF from patient 3, but not in patients I and2. The level of myelin basic protein in CSF and circulat‑

tions indicate that less symptomatic extensive lesions ining adrenocorticotrophic hormone was normal in thesethree patients. Anti‑cardiolipin antibody (ACLA) waswith the virus infection in these patients. However, it
still remains to be elucidated that possible processes,
which result in cerebral white matter abnormal findings

not detected in patient I and 2, but the patient 3 showed

the increased serum ACLA Ievel both as lgG and lgM(ELISA) . The concentration of serum cholestanol was
not increased in patient I and 3 (not tested in patient 2) .

the cerebral white matter may be associated with
HTLV‑ I infection, and suggest that MRI scans may
detect occult diffuse inflammatory change associatedon MRI scans with less correlation with cognitive
impairment (Mirsen et al., 1991; Almkvist et al., 1992;
Tupler et al., 1992), are facilitated by HTLV‑ I infec‑
tion.

DISCUSSION

REFERENCES

Cerebral MRI changes in the present cases werediffuse and extensive, unlike periventricular leukoar‑

1 ) Almkvist, O., Wahlund, L‑O., Andersson‑Lundman, G.,aiosis (caps and lining) and increased T2 signal spots in

Basun, H. and Backman, L. (1992): White‑matterthe deep white matter which are encountered in clinical‑

hyperintensity and neuropsychological functions inly asymptomatic elderly individuals (Shmidt et al.,1992) . Two of these three patients have cerebrovascularrisk factors including cardiac disease in patient 2 andhypertension and serum anticardiolipin antibody, which

would enhance coagulation conditions, in patient 3.
However, the occurrence of ischemia or expanded arte‑riosclerosis (i. e. Binswanger's disease) distributed to all

MRI‑evident lesions can not be predicted from theclinical features in these cases. The diagnoses of central

nervous system disorders including leukodystrophy,
storage diseases, multiple sclerosis, systemic effect of

metabolic abnormalities, and toxins, which show a cere‑bral white matter involvement and may be
asymptomatic in early stages, can be excluded on thebasis of laboratory examinations and clinical features.
Although patient I has spastic paraparesis, the diagnosis

of HAM/TSP is equivocal because of his possible
manifestation of cervical OPLL.

The seropositivity for HTLV‑ I together with adissociation between the minimal cognitive impairment
and remarkable MRI abnormality in the cerebral hemi‑
sphere white matter is the̲common characteristic in our
three patients. Similar cases exhibiting unpredictable
white matter MRI Iesions have previously described inpatients with HAM/TSP (Natori, 1989; Valderrama etal., 1989; Uyama et al., 1991; Koyanagi and lida, 1991),

and a possible correlation between the lesions andcognitive impaiments was discussed (Uyama et al.,dementia and healthy aging. Arch. Seurol., 49, 626‑632
2 ) Furukawa, Y., Une, F. and Osame, M. (1989) : Magnetic

resonance imaging findings of HTLV‑ I ‑associatedmyelopathy. Rinsho Shinkeigaku, 29, 154‑158
3 ) Izumo, S., Usuku, K., Osame, M., Machigashira, K.,

Johnosono, M. and Nakagawa, M. (1989): The neur‑
opathology of HTLV‑ I ‑associated myelopathy inJapan, report of an autopsy case and review of theliterature. In: Roman, G. C., Vernant, J. C. and Osame,M. (eds) : HTLV‑ I and the Nervous System. pp. 261‑267, Alan R. Liss. Inc., New York4 ) Kira, J., Minato, S., Itoyama, Y., Goto, I., Kato, M. andHasuo, K. (1988): Leukoencephalopathy in HTLV‑ I‑associated myelopathy: MRI and EEG data. J. Neurol.
Sci., 87, 221‑232

5 ) Konagaya, M. and lida, M. (1991): A case of HTLV‑ I
associated myelopathy with diffuse white matter lesionof the frontal love and continuous lesion of the pyrami‑

dal tract on cranial MRI. Rinsho Shinkeigaku, 31, 875‑8776 ) Mattson, D. H., McFarlin, D. J., Mora, C. and Zaninovic,

V. (1987): Central‑nervous‑system lesions detected bymagnetic resonance imaging in an HTLV‑ I antibody
positive symptomless individual. Lancet, ii, 49‑50
7 ) Mirsen, T. R., Lee, D. H., Wong, C. J., Diaz, J. F., Fox,
A. J., Hachinski, V, C. and Mersky, H. (1991): Clinicalcorrelates of white‑matter changes on magnetic reso‑
nance imaging scans of the brain. Arch. Neurol., 48,
1015‑1021
8 ) NatQri, N. (1989): Human T‑cell lymphotrophic virus‑type I associated myelopathy with diffuse lesions in1991) . Therefore, the significance of this report involves

cerebral white matter by computed torography andmagnetic resonance irnaging. Nippon Naikagakkai

not only a confirmation of the dissociation between
cognitive functions and MRI findings in HTLV‑ I in‑

9 ) Osame, M. and McArthur, J. C. (1992): Neurologicalfected individuals but also a suggestion that the diffuse

Zasshi, 78, 980‑981

manifestations of infection with human T cell

120

lymphotropic virus type I . In: Asbury, A. K.,
McKhann, G. M. and McDonald, W. I. (eds.): Diseasesof the Nervous System, Clinical Neurobiology, Vol. II,
pp. 1331‑1339, W. B. Saunders Co., Philadelphia10) Shmidt, R., Fazekas, F., Kleinert, G., Offenbacher, H.,
Gindl, K., Payer, F., Freidl, W., Niederkorn, K. andLechner, H. (1992): Magnetic resonance imaging signal
hyperintensities in the deep and subcortical white mat‑ter: a comparative study between stroke pftients andnormal volunteers. Arch. Neurol., 49, 825‑827
1D Tupler, L. A., Coffey, C. E., Logue, P. E., Djang, W. T.

and Fagan, S. M. (1992): Neuropsychological impor‑tance of subcortical white matter hyperintensity. Arch.
Neurol., 49, 1248‑1252

12) Uyama, E., Miyajima, M.. Sugimoto, M., Kawasaki, S.,

lkeda, T. and Araki, S. (1991): A case of HTLV‑ I
associated myelopathy progressed in course over 30
years. Rinsho Shinkeigaku, 31: 301‑305
13) Valderrama, R., Madrid, R. E., Montesinos, C., Merino,

R. R. and Pipia, P. A. (1989): Chronic progressive
myeloneuropathies in an immigrant population in New
York. In: Roman. G. C., Vernant, J. C. and Osame, M.
(eds) : HTLV‑ I and the Nervous System. pp. 185‑194.
Alan R. Liss. Inc., New York

121

Jpn. J. Trop. Med. Hyg., Vol. 22, No. 3 (1994) pp. 121‑124

EPIDEMIOLOGICAL FACTORS AFFECTING
PROGNOSIS OF NEONATAL TETANUS
IN DAKAR, SENEGAL.

PAPA SALIF SOW, BERNARD MARCEL DIOP, MAME AWA FAYE,SALIF BADIANE, AWA MARIE COLL‑SECK.
Received May 6 1994/Accepted July 10 1994

ABSTRACT: To investigate factors affecting prognosis of neonatal tetanus (NNT) , the present study was
conducted in the infectious diseases ward of the Dakar University Teaching Hospital from January 1992 toDecember 1993. One hundred ninety one cases of NNT were collected. According to the questionnaire, themain important epidemiological factors affecting prognosis of NNT are: home delivery, age less than 7days, cutting and dressing the umbilical cord with nonsterile instruments and lack of tetanus imminuzation
during pregnancy.

KEYWORDS: NEONATAL TETANUS, EPIDEMOLOGY, DAKAR.
INTRODUCTION

Neonatal tetanus (NNT) is a major cause of

2. Methods: A questionnaire was used for each case.

The assessement included: age of the neonate, sex,
place of delivery, instruments used for cutting andmorbidity and mortality in developing countries. The

dressing the umbilical cord.

portal of entry is usually the umbilical cord. According

The number of visiting health care center and tetanus
vaccine doses during pregnancy were also obtained fromto WHO(1992), I million chidren contract NNT eachyear, 800,000 of which die. Mortality rates of NNT
varied markedly from country to country, ranging fromO to 70 per 1000 Iive births. Despite implementation ofeach mother.
3. Statistical analysis: Epidemiological data wereanalysed on microcomputers using software for data

the Expanded Programme on Immunization (EPD,

management and epidemiological analyses (EPIlNFO.

NNT remains a great public health problem in the

5, CDC/WHO) . Comparison of frequency of ep‑

Third World.

demiological features in the patients was performed

Previously, the incidence rate of NNT in Dakar was
evaluated at 85 cases per 1000 Iive births (Sow, 1982).

using the chi‑square test. A p value of less than 5% wasconsidered to be statistically significant. The interval
confidence of the relative risk (RR) was calculated with

Therefore, we evaluated the epidemiological factors
affecting prognosis of NNT at the infectious diseases
ward of the Dakar University Teaching hospital by

an interval limit of 95%.

questionnaire.

RESULTSPATIENTS AND METHODS

1. Patients: All patients at the age of O to 28 days

who had clinical findings compatible with NNT (spas‑
ticity, impossibility to feed, risus sardonicus) , wereenrolled in this study at the infectious diseases ward ofDakar University Teaching Hospital from January 1992
to December 1993.

During the study period, 191 cases of NNT wereobserved. One hundred and fifteen (60.2%) were maleand 76 female (39.8%). The lethality rate was high inboth females and males: 56.5% and 52.1% respectively.
However there was no statistical significance in theincidence of death between the two sexes, (p=0.57
Table 1) .

Eighty eight cases (46%) were at an age of less than

Infectious Diseases, Dakar University Teaching Hospital, Fann BP 5035, Senegal.

122

7 days, 91 cases from 8‑14 days and 12 cases more than
15 days. The lethality rate was higher in neonates aged

between O and 7 days (75%) compared with those aged
between 8 and 14 days (40.6%) and aged more than 15days (O%), (p<0.0001 Table 2).
One hundred and fifty cases (79%) were delivered

Table 1. Sex and neonatal tetanus lethality rate.

Sex

Total

Number of deaths (%)

p value

FemaleMale76

43 (56 . 5%)

o . 57115

60 (52 . 1%)

Total

191

103 (53.9%)

at home and 21% at maternity clinics. The lethality rate

was 59.3% in the cases of home delivery versus 34.1% atclinics, p=0.0002 with a relative risk (RR) = 1.77 (1..12<

RR<2.44). (Table 3).

Table 2. Age and neonatal tetanus lethality rate.

Age (days)

Total

Number of deaths (%)

p value< O . OOO1*

0‑7

88

66 (75%)In all cases, portal of entry of Clostridium tetani

8‑14

91

37 (40.6%)

was assumed to be through the umbilical cord cut with

> =15

12

Total

191

nonsterile instruments: razor (34%), kitchen knife(5%), stem (26%). Sterile scissors were used in only
35%. The lethality rate was 59% when nonsterile instru‑
ments were used versus 44% for sterile scissors [p= 0.03

O ( o%)

103* Legend: The rate of lethality of the newborn, aged from 0‑7days, is higher than the other two group aged more than 7
days.

and relative risk (RR) =1.34 (1.02<RR<1.77]

The dressing of the wound stump was made with

Table 3. Place of delivery and neonatal tetanus
lethality rate.

various material and nonsterile materials were used in

Number of deaths (%)

77% of all cases: application of coconut oil, traditional

Place

Total

butter, clay, talcum powder. Multiple and various

Home

150

89

(59 .

methods of tie were used on the umbilical cord: woven
morsel from the cloth of the mother (50%) and thread

Clinic

41

14

(34 .

Total

191

103

pulled from the traditional birth attendant or the mother(43%) . The lethality rate was high when nonsterile ties

were used (62%) compared with that 42% when sterileties were used [p=0.004 with a relative risk (RR) = 1.48

Table 4. Prenatal consultation (PNO and neonatal
tetanus lethality rate.

More than one half of the mothers of these neonates

PNC(62%) had never been to a health care center (HCO
during pregnancy. Only 4.2% had 3 prenatal consulta‑
tions (PNO recommended. The lethality rate was 60%
when mothers did not visit HCC versus O% for thoseattending HCC during pregnancy, (p= 0.00000.. Table 4) .

Lack of adequate tetanus immunization during

a higher rate of lethality of the neonate due to tetanus(65%) . Only 8% had received the 2 doses recommended

by WHO. The incidence of NNT death was influenced

Total

Number of deaths (%)

p value

O PNC
1 PNC
2 PNC
3 PNC

118

78 (66 . 1%)<0.0001*36

14 (38.8%)

29

11

8

O ( O%)

Total

191

Seventy five per cent of the delivery in our study
was done by traditional birth attendants (TBAS) and in
25% by registered midwives. The lethality rate was 61.

2% for TBAS and 31% for registered midwives, (p=0.
O002) .

(37.9%)

103* Legend: The lethality rate is higher when mothers did not
visit the health care center during pregnancy.

Table 5. Tetanus toxoid immunization during pregnancy
and neonatal tetanus lethality rate.

Number of

Tetanus toxoid

Total

O dose140

85 (60 . 1%)
13 (36 . 1%)

by the vaccinal status of the mother, (p=0.0001 Table5) .

o . o002 ** Legend: The lethality rate is higher in cases delivered at homethan that at maternity clinic.

(1.12 < RR < 1.95]

pregnancy is frequently seen (73%) in our study. Whenmothers had not been immunized, it was associated with

3%)
1%)

p value

1 dose36

2 doses

15

Total

191

deaths (%)

p valueO . OOOI *5 (33.3%)

103* Legend: The lethality rate is higher when mothers had notreceived tetanus vaccine; no difference is found by frequencyof immunization.

123

Photo 1: Case with neonatal tetanus showing spasticity

Photo 2: The infected umbilical cord by the way of cutting

and dressing is usually the portal of entry ofand risus sardonicus,

Clostrldium letani.

DISCUSSION

and rural areas of developing countries, Iack of healtheducation, availibility of health services, environmental

NNT is a comman disease in the infectious diseases
ward of the Dakar University Teaching hospital. Previ‑
ously, the predominance of NNT among neonatal infec‑
tions in Dakar had been evaluated at 85% Coll et al.,1986). It also had an important place among neonatal
infections during the study period. NNT formed 96% of
all serious neonatal infections, including bacterial men
ingitis (3 cases), pneumaniae (2 cases) and septicemia

and socioculturai behaviour are factors contributing to
the marbidity of NNT. A case ‑ contral study conductedin a rural area in Senegal, showed that contamination of(1 case) . According to WHO (1992), NNT represents
irom 40 to 80% of all admitted neonatal iufections indeveloping countries, such as Africa and SouthEast

umbilical cord for ail cases, bec'ause these babies did not

Asia.

umbilical cord in Dakar (Alihonou, 1969; Sow, 1982; Feil,In our study, the lethali,'ty rate was 53.9%. High lethality

1987). In developing countries, the portal of entry of
Clostridium tetani is usually the umbilical cord, indue to NNT is also found in other African countries, inTogo with a rate af 71% (Grunitzky et al., (1991), in
Nigeria with a rate of 68% (Ergie, 1992) and in Ethiopia

the hands and careless dressing by TBAS were signifi‑
cantly associated with a high incidence of NNT (Leroy
and Garenne, 1991) .
Although we did not try to isolate the bacteria, theportal of entry of Clostridium tetani was probably thehave any other lesion where the pathogen could enter. It

was demonstrated that 95% of NNT had an infected

Burkina faso (Tall, 1991), Togo (Grunitzky, 1991),
Bangladesh (Hladly et ai., 1992) and in Ethiopia

with a rate of 40% (Alemu, 1993). Commonly, the highlethality due to NNT in Africa can be explained by(Alemu, 1993) .

multiple factors, such as, severity of tetanus in neonates*

induced a higher mortality in the present study, because
hygenic principles are not executed. From data analysis,
it is suggested that the main source of Clostridium tetani

consultation‑delay and lack of adequate intensive careunits.

We clearly revealed that home delivery by TBASIn the present study, we found that the majority ofour patients were at age of less than two weeks with amean age of 7 days, at admission. The lethality rate ismay be the hands of the TBAs. The use of a nonsterile
instrument for cutting the umbilical cord and the un‑very high during this period. According to the literature,

make for heavy contamination by bacteria resulting indevelopment of serious infection causing higher leth‑a shot incubation period is a bad prognosis factor(Ferron, 1969; Feil, 1987; Whiteman et al., 1992) intetanus.

sanitary method of dressing the wound stump mightaiity.

Lack of immunization of tetanus toxoid during

The majority of our NNT cases (71%) were trans‑ferred from the suburban and rural areas. Previously,
the high incidence of NNT in these locations has beenpregnancy is also a very important risk factor for deathemphasized by several authors (Huault, 1964; Alihonou,

tetanus vaccine (Gasse, 1987). Eight per cent of the
NNT cases in the present study were babies delivered

1969; Sow, 1982; Galazka, 1984; Cliff, 1985). In suburban

due to NNT in our study. Only ll% of the women in
Airica had received the recommended two doses of

124

by the mothers who had received two doses of tetanus
toxoid immunization during pregnancy. A case‑control

nus. Lancet, 1, 8380, 789‑790.
10) Gasse, F. (1987) : Neonatal tetanus elimination in Africa,

study conducted in rural Bangladesh found that the risk

Semin. Intern. sur les vaccinations, Niamey, 30‑31

of NNT was not reduced by receiving two doses ofjanvier, Collection Fondation Merieux.

tetanus toxoid (TT2) (Hlady et al.,1992). They esti‑
mated the efficacy of TT2 at 45% and emphasized theneed for improved quality of tetanus vaccine in develop‑
ing countries.

The present study reveals that these multiple
epidemiological factors during pregnancy and at deliv‑
ery contributes to a high lethality rate of NNT.
Health facilities and health education must be im‑proved in the Third World. Acceleration and promotionof tetanus toxoid immunization of all women of child‑bearing age are very inportant priorities. Training of
TBAS for proper obstetric care, coupled with continuous

supportive supervision, is recommended in order tosuccessfully execute NNT elimination in developing
countries.

11) Grunitzky‑Bekele, 'M., Apetoh, A. (1991): Neonatal
tetanus in Lome‑Tonkin Togo, Bull. Soc. Pathol. Exot.,
84, 540‑549.

12) Hinen, A.E. et al. (1969): Neonatal tetanus‑report acase with recovery, Am. Dis. Child., 39, 560‑562.
13) Hlady, W., Bennett, J.V., Sawadi, A.R.. Begun, J., Hafez,
A., Tarafdar, A., Boring, J. (1992): Neonatal tetanus inrural Bangladesh: risk factors and toxoid efficacy, Am.
J. Public Health, 82, 135‑139.

14) Huault, G. (1964): Neonatal tetanus, Medical Thesis,
Paris, no 78.

15) Leroy, O., Garenne, M. (1991): Risk factors of neonatal
tetanus in a rural area in Senegal. Int. J. Epidemiol., 20,

521‑526.

16) Sirnol, ,J. (1964): Treatment of neonatal tetanus in
Africa. Med. Trop. 24, 573‑575.
17) Sow, A. (1982): Neonatal tetanus in Dakar: New data,Afri. Med., 21, 13‑22.
18) Tall. F., Prazuck, T., Roisin, A., Sanou, J. (1991): Risk

ACKNOWLEDGEMENTS
The authors would like to express their sincere andheartfelt thanks to Doctor Naoto Rikitomi for his criti‑
cal and his helpful suggestions reading of this manu‑
script. We also express our thanks to the Department ofInternal Medicine of the Institute of Tropical Medicine,
Nagasaki University, for supporting this manuscript.

REFERENCES
1 ) Alemu,W. (1993): Neonatal tetanus survery, north andsouth Omo administrative regions, Ethiopia, Ethiop.
Med. J., 31, 99‑107.

2 ) Alihonou, M.E. (1969): Neonatal tetanus in tropicalareas. Medical Thesis, Dakar, no 7.
3 ) Badiane, S., Coll‑Seck, A.M., Diop, B.M., Feil, O., Sow,

P. S. ( 199 1 ) : Neonatal tetanus i n Da k a r:
Epidemiological and clinical aspects, Bull. Soc. Pathol.
Exot., 84, 550‑557.
4 ) Cliff, J. (1985): Neonatal tetanus in Maputo: Preventive

measures. Centr. Afr. Med. J., 31, 32‑35.
5 ) Coll, A.M., Sow, P.S., Diop Mar, l., Sow, A. (1986) :Neonatal infections in Dakar, Med. Mal. Infect. 4, 127‑130.

6 ) Ergie, C. (1992): Neonatal tetanus estimates of mortal‑
ity derived from a cluster survey in Northern Nigeria, J.
Infect., 25, 133‑137.

7 ) Feil, O.A. (1987): Clinical presentation of neonatal
tetanus in Dakar, Medical Thesis, no 22.
8 ) Ferron, E.L. (1969) : Neonatal tetanus, Medical Thesis,
Bordeaux, no 7.
9 ) Galazka, A., Cook, R. (1984): Preventing neonatal teta‑factors for Neonatal tetanus in Western Burkina faso:case‑control study, Bull. Soc. Pathol. Exot., 84, 558‑561.
19) Whitrnan, C., Belgharbi, L., Gasse, F., Torel, C., Mattei,

V., Zoffman, H. (1992): Progress towards the global
elimination of neonatal tetanus, World Health Stat. Q.,
45, 248‑256.

125

Jpn. J. Trop. Med Hyg Vol 22 No 3 (1994) pp. 125‑127

STOOL ANALYSIS OF PEDIATRIC DIARRHEAIN SURABAYA, INDONESIANoBORU NAKASONE*, MASAAKI IWANAGA ,', EDDY BAGUS WASITO'PITONO SOEPARTO', and I. GUSTI NYOMAN GDE RANUH*
Received May 13 1994/Accepted 15 July 1994

Abstract: Forty seven stool specimens of pediatric diarrhea in Surabaya, Indonesia during the period from

July to August 1993 were studied. The isolation rates of enteropathogens were as follows: 47% forenteropathogenic Escherichia coli (EPEO , 11% for Vibrio cholerae. 9% for Shigella spp, 21% for Rota virus
and so on. The mixed infection found in 8 cases out of 35. A pH of the diarrheal stools was variable fromcase to case. The concentration of lysozyme and protease in diarrheal stools were higher than those innormal control stools, although the total protein content in diarrheal stools was lower than that in control
stools.

past decade. However, a microbiological study for

Biken method for LT (4), and heat‑stable enterotoxin(ST) of E. coli by using the ST detection Kit (COLISTEIA: Denka Seiken Co., Tokyo, Japan) . Rotavirus was
examined by using the Rotavirus detection kit (Denka
Seiken Co., Tokyo, Japan) .
Biochemical examination: The stool samples werediarrheal disease should be continued, because the isola‑

centrifuged at 10,000 rpm for 10 min in Eppendrof tubes.

tion frequency of the enteropathogens differs from place
to place, from year to year, and the patterns of their

Each supernatant was tested for pH, total protein con‑tents, and the activities of lysozyme and protease. The
control stools were diluted roughly ten times in normal
saline solution and centrifuged as described above, andthe supernatants being used for the biochemical ana‑lyses. Protein concentrations were determined by using
the Bio‑Rad protein assay kit (Bio‑Rad Lab., Calif., U.

INTRODUCTION
The oral rehydration solution (ORS) therapy
promoted by Primary Health Care activities has lead todecreasing the mortality rate of diarrheal diseases in the

drug sensitivity are changing. In this communication,
the enteropathogens and biochemical analysis in thestools of pediatric diarrhea in Surabaya, Indonesia aredescribed.

MATERIALS AND METHODSPatients and stools: A total of 47 patients aged

from 5 to 24 months at Dr. Soetomo Hospital (Sur‑abaya, Indonesia) were exarnined during the period
from July to August in 1993. The stool samples werecollected in plastic containers, and subjected to exami‑

S. A.) with bovine serum albumin as the., standard.
Lysozyme activity was bioassayed using Micrococcus
luteus NCTC2665 as a substrate. Crystallized hen‑egg

white lysozyme was used as the standard. Total
proteolytic activity was assayed on skim milk agar
plates as described by Honda et al. (3) with protease K
as the standard.

nation within 2 hours after sampling. The stools from 38

healthy children under 2 years of age were used ascontrols.

RESULTS

Microbiological examinations: The isolation of

Isolation frequency: Enteropathogenic bacteria

Escherichia spp., S lmonella spp., Shigella spp., Vibrio

were isolated from 35 cases out of 47 examined (74%) .

spp. and Aeromonas spp. were carried out from all
diarrheal stools as previously reported (5). Heat‑labile

Enteropathogenic E. coli (EPEO predominated path‑ogens (Table D. Enterotoxigenic E. coli (ETEO wasenterotoxin (LT) of E. coli was tested by using theisolated from two cases. They produced ST only. Rota '1 Department of Bacteriology and 2 Research Institute of Comprehensive Medicine, University of the Ryukyus School of Medicine.
207 Uehara, Nishihara, Okinawa 903‑01, Japan. 3 Department of Microbiology, 4 Department of Pediatrics, and 5 Tropical Disease
Research Center, Airlangga University, Dharumahusada 47, Surabaya, Indonesia.

126

Table I Isolation Frequency
of Enteropathogens

No. examined

47

Pathogen detected

35(74%)
22(47%)
2( 4%)
4( 9%)
5(11%)
1( 2%)
10(21%)EPEC
ETEC
Shigella

V. cholerae

Aeromonas
Rota virus
Pathogen not detected

12 (26%)

Table 2Biochemical analysis of the stools
Grou p

IV

I

II

pH

5.2il.O5.9 1.5

5.7 1.4

5.1 0.7

Total protein

193:!:90

523

437

928

3.3:!:4.3

3.1i6.5

4.1 :2.8

45 . 5

III

430

399

805

(mcglgr)

Lysozyme(mcglgr)Protease(mcglgr)

182

3.1i5.6

0.2 0.2

33 . 7 i 154 . 4

1.2 1.4

I : diarrhea, pathogen detected
II : diarrhea, pathogen not detected
III : diarrhea, ( I + 11 )IV : control stools

Numerals indicate the mean value standard deviation.

virus ranked second in the isolated pathogens. The

Protozoa, etc., or may be due to other reasons including

isolation rates of Shigella spp. and V. cholerae Ol wereuse of antibiotics.

9% (4147) and 11% (5/47), respectively. From 8 cases,
more than 2 pathogens were isolated.
Biochemical analysis of stools: Since biochemical
data were so variable from case to case, the patients

Eight (17%) out of 47 cases excreted two or threepathogens in the same stool sample. The mixed infec‑
tion in children with diarrhea is commonly seen else‑

were classified into 3 groups as follows (Table 2) ; Group

which pathogen is responsible for the illness.

where (1, 5, 6, 8). In such cases, it is difficult to decide

1 includes diarrheal patients with enteropathogens

Rota virus was detected at about 20%. The highdetected, group 11 includes diarrheal patients without
enteropathogen, and group 111 includes all cases with orwithout detected pathogens. Healthy control children
were classified as group IV. The data were expressed as
the mean values of each group and their standard devia‑
tions (Table 2). The pH of the stools revealed a great
variety. The activities of lysozyme and protease wereisolation rate of this virus in Surabaya was previously
reported by lwanaga et al. (5), and also in the otherhigher in diarrheal stools than in normal stools, although

the total protein contents in normal stools were higher
than those in diarrheal stools.

DISCUSSION

countries in Asia (1, 6, 8) . Further investigations areneeded to collect more detailed information on it.
The relationship between enteropathogens isolated

and biochemical compositions of the stool was notdetermined in the present study because of the small
number of samples. However, its relation must be very
important to understand pathophysiology of the diarr‑
heal diseases. The inversion of total protein contents
and enzyme activities between diarrheal and normalstools may be explained in part by following specula‑

The enteropathogens were detected in 74% of 47

tions; 1) A high fluid contents in diarrheal stools may
reduce the protein contents, 2) An inflammation caused

diarrheal cases. This detection rate seems to be about

by enteropathogens may lead to a high enzyme activ‑

the same with the results of other studies reported (1, 5,

ities. However, further investigations will be need tocorrect the speculations. Comparative studies of the6, 8). In agreement with another report (5), we foundthat EPEC was the bacterial agent most frequently
isolated from children with diarrhea in this area. The
recovery rate of ETEC (4%) was lower than reported in
China(20%) (6) and Philippines (17.8%) (1). The rateof isolation of Shigella spp. (9%) are similar to thatreported in India (6%) (8) and Philippines (11.6%) (1),chemical composition of stools between diarrheas with asingle pathogen and normal stools, or between diarrheas

caused by each pathogen will elucidate the specificresponse of the host against each specific pathogen.
Vibrio cholerae 0139 which had caused an epidemicin India and Bangladesh (2, 7) was not isolated in thisbut higher than that reported in China (3%) (6).

research. However, since the organisms are spreading

Diarrhea in 12 cases without detection of enteropath‑ogens may be due to other pathogens, such as

to the neighboring countries, we should carefully moni‑
tor the enteropathogenic bacteria causing diarrhea inCampylobacter spp., Bacteroides spp., Clostridia spp.,

this areas.

127

REFERENCES
1 ) Adkind, H. J., Escamilla, J., Santiago, L. T., Ranoa, C.,

Escheverria, P. and Cross, J. H. (1987) : Two‑year
survey of etiologic agents of diarrheal disease at San
Lazaro Hospital, Manila, Republic of the Philippines. J.
Clin. Microbiol. 25: 1143‑1147.

2 ) Albert M. J., Ansaruzzaman, M., Bardhan. P. K., Faru‑
que, A. S. G., Faruque, S. M., Islam M. A.. Siddique, A.
K., Yunus, M. D. and Zaman, K. (1993) : Large epidemicof cholera‑like disease in Bangladesh caused by Vibrio
cholerae 0139 synonym Bengal. Lancet. 342: 387‑390.
3 ) Honda, T., Booth, B. A,, Boesman‑Finkelstein, M. and
Finkelstein, R. A. (1987): Comparative study of Vibrio
cholerae non‑OI protease and soluble hemagglutinin withthose of Vibrio cholerae O1. Infect. Immun. 55: 451‑454.

4 ) Honda, T., Taga, S.. Takeda, Y. and Miwatani. T.
(1981) : Modified Eleck test for detection of heat‑labile
enterotoxin of Enterotoxigenic Escherichia coli. J. Clin.
Microbiol. 13: 1‑5.

5 ) Iwanaga. M., Nakasone, N., Nakamura, S., Wasito, E.
B., Soeparto, P., Sudarmo, S. M., Soewandojo, E. and
Ranuh G. N. G. (1993): Etiologic agents of diarrheal
diseases in Surabaya, Indonesia. Jpn. J. Trop. Med.
Hyg., 21: 143‑147.
6 ) Kain, K. C.. Barteluk, R. L., Kelly, M. T., Xin, H., Hua,
G. D., Yuan, G., Proctor, E. M., Byrne, S. and Stiver, H.

G. (1991): Etiology of childhood diarrhea in Beijing,
China. J. Clin. Microbiol. 29: 90‑95.

7 ) Ramamurthy, T., Garg. S., Sharma, R.. Bhattacharya, S.
K., Nair, G. B.. Shimada, T., Takeda, T., Karasawa. T.,

Kurazano, H., Pal, A. and Takeda, Y. (1993): Emer‑
gence of novel strain of Vibrio cholerae with epidemic
potential in southern and eastern India. Lancet 341: 703‑704.

8 ) Sen, D., Saha, M. R., Niyogi, S. K., Balakrish Nair, G.,
De, S. P., Datta, P., Datta, D.. Pal, S. C., Bose, R., andRoychowdhury, J. (1983) : Aetiological studies on hospi‑
tal in‑patients with acute diarrhea in Calcutta. Trans.

Roy. Soc. Trop. Med. Hyg. 77: 212‑214.

129

Jpn. J. Trop. Med. Hyg., Vol. 22, No. 3 (1994) pp. 129‑131

A COMPARATIVE STUDY ON STAPHYLOCOCCUS A UREUS
ISOLATED IN LAO PDR AND IN JAPANNAOMI HIGA*, NoIKASEUMSY SITHIVONG', and MASAAKI IWANAGA*,

3

Received June 15 1994/Accepted July 28 1994

During the past decade in Japan, methicillin resis‑
tant Staphylococcus aureus (MRSA) has been recognized

Drug sensitivity patterns of the isolates from Laosand Japan were clearly different from each other. MIC90

as one of the major pathogens for nosocomialof EM was 0.4 pg/ml in Lao isolates but 100 pglml or
more in Japanese isolates. Actually Japanese isolates
revealed 2 peaks of susceptibility against EM (Fig. 1‑A) . TC was less effective to Lao isolates. MICs of TC
against 58% of Lao isolates and 25% of Japanese iso‑infection5, 6), and the lethal cases due to MRSA wereoccasionally seen'). Increasing of its isolation frequency

was coincident with increasing use of third‑generation
cephem‑antibiotics. Therefore, the careful use of these

antibiotics is now recommended. MRSA may rarely beisolated in the area such as Lao PDR where the third
generation cephems are not used. In this communica‑
tion, the strains of S. aureus isolated in Lao PDR aredescribed with reference to their drug sensitivities andcoagulase types comparing to those in Japan.
Staphylococcus aureus isolated at Mahosoto Hospi‑

lates were 12.5 pg/ml or more. (Fig. 1‑B). MICs of
ABPC showed a pattern of normal distribution with thefrequent MIC of 3.13 pg/ml in Lao strains and 25 pglml
in Japanese strains (Fig. 1‑O . A11 Lao isolates were
inhibited at 0.8 pg/ml or less of CFDN, whereas only

37% of Japanese isolates. Thirty‑eight per cent ofJapanese isolates were resistant to 100 pglml of CFDN

tal, Vientiane, Laos and at the Ryukyu University(Fig. l‑D) . All Lao isolates were inhibited at 6.25 pgl

Hospital. Okinawa, Japan were used. The 54 strains of

ml or less of DMPPC, but 63% of Japanese isolates were
not inhibited at 100 pglml of DMPPC (Fig. 1‑E) .
The most frequent antigen type of coagulase in Lao
isolates was type 5 (54%), and in Japanese isolates, it

Laos were isolated in 1993, and 95 strains of Japan wereisolated in 1992.

Minirnum inhibitory concentrations (MICs) of
Erythromycin (EM), Tetracycline (TO , Ampicillin(ABPO, Cefdinir (CFDN), and Methicillin (DMPPO
were determined by plate dilution method. Two folddilution series of each drug in heart infusion agar (HIA)

was prepared with the drug concentration ranging from100 to 0.2 pg/ml. The concentration of NaCl in methicil‑
lin containing HIA plates was adjusted at 4%. Culturesof the isolates in heart infusion broth at 37'C for 6hr
were diluted I to 10 with normal saline solution (ca. 107/mD , and were inoculated by using Microplanter (Sa‑kuma Co. model MITP #00257) on the drug containing
HIA plates including a control plates without drug.
MICs of each drug were evaluated after 24hr incubation
at 37'C.

Antigenic types of coagulase produced by the iso‑lates were examined by neutralization test using"Coagulase Typing Immune Sera Kit" (Denka SeikenCo., Tokyo) . Coagulation inhibition by adding anti‑sera
was examined using plastic microdilution plates7).

was type 2 (53%). There were 11% of type 2 in Lao
isolates and there were only 4% of type 5 in Japaneseisolates (Table 1).

Table 1. Cagulase typing

Laos

type

Ja pan

o%

1

1%

50

6

11%
11%

53%
19%

2

4%

2

5

29

54%

4

6

o

o%

1

7

8

15%

13

14%

8

1

o

NT

2

2%
4%

6

o%
6%

total

54

100%

95

100%

1

o

2

6

3
4

18

2%
4%
1%

In Lao PDR, Mahosoto Hospital is the greatest
hospital in the viewpoints of its size and function.
Antibiotics being used over there are penicillin‑G, amox‑

Department of Bacteriology* and Research Center of Comprehenisive Medicine', Faculty of Medicine,
University of the Ryukyus; 207 Uehara, Nishihara, Okinawa 903‑01, Japan.
National Institute of Hygiene and Epidemiology', Vientiane, People's Democratic Republic of Laos.

130

EM

1・A

90

CFDN

1・D

7

％

％

80

︵
︵う
︵﹂︵
54
2︹
10

70

60
50

葡3020100
0．2≧

0，2

0．4

0，8

1，6

3．136，2512．5．25

50 100100＜

0，2≧0，20，40．8

1．63．136．2512，5

25

50

μ砂ml

μ9／ml

η

TC

1−B

80

100100＜

DMPPC

1−E

1島

1／1▼πrし

60

D
％

％

70

50

60

⑳

50

30
20

㈹3020mO

1000，2≧0，2

0，4

0，8

1，6

3，136，2512，5

2550100100ぐ

0，2≧0，2

0，4

0．8

1．6

3．136，2512，5 25

μ創m1

ABPC
A口rし

︷J

1・C

1し

％

4つ﹂つ乙
1
、
0，2≧0，20，40．81，63．136．2512，525

50100100＜

μガml

Fig．1

MIC

of

囮Laos

A：Erithromycin，B：Tetracycline，C：Ampici in，D：Cefdinir，E：Methicillin

画Japan

50

100

100＜

，曜ml

131

icillin, tetracycline, chloramphenicol, gentamicin,

Kosyu Eisei Zasshi, 40, 235‑239

nalidixic acid, and erythrom cin. Among them, ampicil‑

7 . Tajima, Y., Nagasawa, Z., Tanabe, I., Yamada. H.,

lin and tetracycline are most frequently used. Reflecting

Kusaba, K. and Tadano, J. (1992) : An Improved
Method for the Serotyping of Free Coagulase fromthis background, considerable number of S. aureus resis‑

tant to these two drugs were isolated. Erythromycin isless frequently used in Laos because of its price, andthere were very few resistant isolates. It is noteworthy
that there was no MRSA in the examined isolates from
Lao. However, it is no wonder because of the antibiotics

consumption pattern in Laos. The third‑generation
cephems, which are closely related to the developmentof MRSAl, 3), might have never been used over there.
The frequency of MRSA at Ryukyu University Hospitalis in an average level of all Japan2). It is well recognizedthat nosocomial infection due to MRSA is a serious
problem in the present Japan, however, there is no
problem of this resistant organism in Laos. Reviewing
the drug sensitivity pattern in Laos and Japan, we haveto consider the proper use of antibiotics.

Coagulase type of S. aureus can be used for an
epidemiological study. The most frequent coagulase
type in Laos and Japan was type 5 (54%) and type 2(53%), respectively. While, the frequency of type 5 inJapan was only 4% and type 2 in Laos was 11%. It is notknown what the type 5 means, but type 2 is closely
related to MRSA. The relation between coagulase typeand disease specificity should be considered.

ref erences
1 . Watanabe, A., Ohizumi, K., Aonuma, S., Ono, R., Honda,
Y., Tokue, Y., Kitamura, N., Shoji, S. and Konno, K.
(1989) : Studies on Multiple‑Resistant Staphylococcus

aureus (IV); Difference in Incidence of Methicillin‑Resistant Staphylococcus aureus (MRSA) among Insti‑
tutions. Departments and Specimens, Chemotheraphy,
37, 125‑130
2. Igari, J., Takamine, F. and Imamura, S. (1990) : Present
Status of Methicillin‑Resistant Staphylococcus aureus
ahd Susceptibility to Anitimicrobial Agents in 6 Hospi‑
tals in Okinawa, Jpn. J. Clin. Pathol., 38, 975‑982
3 . Hori, K., Yura, J., Shinagawa, N., Sakurai, S., Mashita,
K. and Mizuno, A. (1989) : Postoperative Enserocolitis

and the Current Status of MRSA Enterocolitis ‑The
Result of a Questionnaire Survey in Japan‑, Kansen‑syougaku Zasshi, 63, 701‑707

4 . Wada, K.. Suzuki, N.. Kawashima, T., Tsukada, H.,
Ozaki, K. and Arakawa, M. (1992) : A Clinical Study of
Bacteremia for the Recent Fifteen Years, Kansenshoga‑
ku Zasshi, 66, 620‑627

5 . Kusano, N. and Nakasone, I. (1990) :Nosocomial

Infection with MRSA. Jpn. J. Clin. Pathol., 38, 990‑997

6 . Yokota, K. (1993) : Nosocomial infecti,ons, Nippon

Staphylococcus aureus. Microbiol. Immunol., 36, 1233‑
1237

Jpn. J. Tro p. Med. Hyg., Vol. 22, No. 3 (1994)

133

pp. 133‑137

THE ROLE OF TUBULAR PROJECTIONSOF PNEUMOCYSTIS CARINII IN
PATHOGENICITY OF P. CARINII PNEUMONIATSUNEZO SHIOTA, YOSHIHARU SHIMADA, HIROSHI KURIMOTO,HIROSHI OIKAWA and NAOKI ARIZONO
Received May 20 1994/Accepted 22 July 1994

Abstract: The aim of this research is to clarify the role of tubular projections of Pneumocystis carinii in P.

carinii pneumonia. Rats, mice and cats with P. carinii infections were investigated by electron microscopy
and semithin section light‑microscopy. We found that the tubular projections adhered closely to the type I
alveolar epithelial cells in the same manner as the body of trophozoites attached to the type I epithelium.
Nurnerous tubular projections were observed between the type I epitheliurn and small clusters of troph‑ozoites, and some of these projections adhered closely to the type I epithelium. In the heavily infected
alveoli, many tubular projections were found around the trophozoites. These projections are likely to link
the organisms, creating a big mass of organisms too big to be discharged from the lung by the surfactant
alveolar clearance mechanism. We think that the tubular projections may play a role in the adherence ofP. carinii to the type I epitheliurn as well as in formation of large masses of these organisms that cannot
be cleared from alveoli. This can then give rise to an increasingly severe respiratory failure.

INTRODUCTION

are one of the specialized structures which allow P.
carinii to fix themselves to type I epithelial cells of

The d. evelopment of Pneumocystis carinii pneumo‑

alveoli that expand and contract during respiration.

nia is significantly affected by the host defense mecha‑
nisms. It is generally considered that impaired cellular

Furthermore, the projections around P. carinii may also

immunity is more important than impaired humoral

carinii that hinders respiratory air exchange.

adhere to each other giving rise to a big mass of P.

immunity in predisposing to P. carinii infection. On theother hand, the parasitic behavior of P. carinii is also
thought to be critical for the manifestation of its path‑

MATERIALS AND METHODS

ogenicity. P. carinii has been reported to be adherent to

The methods of provoking pulrnonary alveolus fill‑type I alveolar epithelial cells and form a significantly

ing with P. carinii in cortisone‑treated rats, convention‑

large mass in the alveoli. This parasitic behavior seems

ally raised nude mice and corticosteroid‑treated cats

to be related to interference with gas diffusion andhave been described (Frenkel et al., 1966; Shiota et al.,failure of ventilation. The pellicle of P. carinii troph‑

1986, 1990) . For the electron microscopic inventigations

ozoite carries tubular projections, called tubular expan‑

the lungs were removed and cut into srnall pieces. These
tissue fragments were then put in a specially designed

sions, that show the same structure as the pellicle fromwhich they arise (Vavra and Kucera, 1970; Ham et al.,
1971; Vossen et al., 1978). Freeze‑fracture replication

plastic syringe containing 2.5% glutaraldehyde instudies of P. carinii revealed that the projection could befrom the alveoli by negative pressure produced byclassified morphologically into four types as follows:withdrawal of the plunger (Ebe et al., 1968; Takeuchi,1980). The tissues were irnmersed in the same fixative

tubular, Iobopodia, branching and beaded structures(Yoshikawa et al., 1987). However, the role of tubular
projections in the pathogenicity of P. carinii pneumonia
is not clear.

In this study, we show that the tubular projections

cacodylate buffer (0.05M, pH 7.4) , and air was removed

for 3 hours at 4'C and washed with the same buffer
containing 8.2% sucrose. Then the tissues were dehy‑
drated through a series of acetone and embedded in
epoxy resin. Ultrathin sections were cut with a diamond

Department of Medical Zoology, Kyoto Prefectural University of Medicine, Kawaramachi Hiroko ji, Kamikyoku, Kyoto 602, Japan

134

A /

/j( i# ' :

""' i
;‑*1'

^*' ^

* * Fi' !

*" ' "

'

;i

'

1"

.' "

* ;
#/ "!

lf

*

'

¥..

,

e(

; ' '''< '*:'fr !

); *f;*<‑=' * "'* :"""‑‑ ;' ;=' = =

;i

:

' ̲

' '

;'*:i*' ix̲{':!'** ** ‑ "i' ":':*;;::' ',2 aly infected alveoli from the lung of a rat (after five

weeks of corticosteroid treatment) . In this figure,
about 80 trophozoites(T) and 2 cysts(C) are obser‑

ved along the surface of epithelial cell. A, airspace of alveoli; Cp, capillary; Am, alveolar macro‑ll'; "='

1;' ]:;tr2̲. 'b : ':{ ;'/ ; #;? >"'=;=

Figure 1 JB4 plastic‑embedded I ,um thick section of light‑:' *;": ;** '< *. ̲ ̲:i:1

71

=+*

‑'<=

: ̲'

if':=i':i""" ' '"̲*i

Figure 2a‑b Cat' cortiCosteroid treated for fi¥re months'
2a: A fe v tubular proiections (arrows) are foundat the periPheral attachment surface (̲)f a small
trophozoite (T) adhering to the respirator}' mem‑brane' 2b: Higher magnigication of the area in thesquare in Figure 2a shows that the trophozoite isphage; * , a small cluster of P. cariylii; Scale bar, 10

attached to the respiratorv membrane' A' air,etm. (Giemsa).

space of alveolus; p' pelliCle of the trophozoite; EP'cytoplasm of type I epithelium; B' basement
membrane; Ed' endothelial cell; RbC' red bloodcell' SCale bar' 100 nm'

knife and placed on uncoated copper drids. The ultrath‑in sections were stained with uranyl acetate and lead!: (';

citrate, and observed under a JEOL 100S electronA

microscope. The procedures for light microscopic inves‑tigations of Giemsa stained I Jclm sections embedded in
JB‑4 plastic were briefly as follows. The sections were
stained vith Giemsa (diluted 20 times with pH 7.2 PBS) .
More than three hours staining in an incubator at 37'C
was required to obtain satisfactory results because thesections were so thin (Shiota, 1984).

:

'

':'

iti ;'; ' :: ' :' '

'‑'}

:r

" t' ':'"' / ss :" '1';:‑ ' <

*' '
/ l{.

"::

;:?f

:

Lightly infected alveoli:

Lightly infected alveoli were examined in I /Im
section embedded in JB‑4 plastic and stained with
Giemsa. As shown in Figure 1, the alveolar septa were
almost normal, and contained much air. The alveolar
capillary membrane (respiratory membrane) and blood
capillaries were well preserved. Trophozoites and cysts
were found to be closely attached to the type I epith‑

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" i ' s' :"^.. / :;<i 'Flgure 3a‑b Nude mouse' five months old, conventionallyraised' 3a: Many tubular projections developing
between a smau cluster of trophozoites (T) andthe respiratory membrane' 3b: Higher magnifica‑tion of the area in the square of Figure 3a showing
some of the projections (arrows) adhered closely

to the type I epithehum'A, air space of alveolus;

elium. Measurements of the respiratory membrane in

Rbc, red blood cell; Cp, capillary; Ep, cytoplasm of

Figure I showed that about 20% of the entire membrane
was covered with the organisms.

type I epithehum; B' basement membrane; Ed,
endothelial ceu' Scale bar' lOO nm.

In TEM sections, fine structures of P. carinii weresimilar to those reported previously (Vavra and Kucera,

were found peripheral to the attachment surface of1970). A membrane to membrane attachment was

trophozoites (Figure 2) . Small cllusters of trophozoites

observed between the trophozoite of P. carinii and type

frequently showed much better developed tubular pro‑

I epithelium (Figure 2). A few tubular projections

jections than the solitary single trophozoite. Tangles of

135

T *
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/

* ,"
i'l ;,?s

Figure 4 Cat, corticosteroid treated for five months. Sometubular projections (arrows) invaginated in thetype I epithelium. T, trophozoite; Ep, cytoplasmof type I epithelium; B, basement membrane; Ed,

Figure 5 Cat, corticosteroid treated for five months. A few
tubular projections (arrow) are found o̲ n the sideof the cyst (C) adhering to the respiratory mem‑endothelial cell; Rbc, red blood cell. Scale bar, 100

tubular projections is seen next to the cyst. Rbc,
red blood cell. Scale bar, I Jtlm.

brane. An empty trophozoite (ET) with manynm.
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Figure 6 Rat, corticosteroid treated for five weeks. Manytr( )phozoites (T) and cysts (O are forming a big
mass of organisms in heavily infected alveoli. The

Figure 7 Nude mouse, five months old, conventionallyalveolar air space (A) was almost completelyaround the trophozoites filling the heavily infectedoccupied with the clumped trophozoites and cysts.
Cp, capillary. Scale bar, 15 pm. (Giemsa).

alveolus. Scale bar, I pm.

raised. Many tubular projections are foundnumerous projections were observed between the troph‑ozoites and type I epithelium (Figure 3a). Some of the
projections adhered closely to the type I epithelium inthe same manner as the trophozoites attached to its
surface. That is, the surfaces of the organism and theclosely to the type I epithelium in the same manner as

host cells ¥vere closely opposed without fusion of the cell

membranes (Figure 3b). Some tubular projections

Heavily infected alveoli:
In the JB‑4 plastic embedded sections, many alveoli

invaginated into the type I epithelium so that thewere filled with trophozoites, cysts, debris of the hostepithelial cells became

projections were also found peripheral to cysts adhering

cells and occasionally with foamy macrophage (Figure6). In TElvl sections, many tubular projections wereto the type I epithelium (Figure 5). The cyst adhered

found around the many trophozoites filling the alveolus

vider (Figure 4). A fe¥v tubular

the single trophozoite shown in Figure 2a. An empty
trophozoite with many tubular projections was often
seen beside such cysts (Figure 5) .

136

(Figure 7). These tubules showed some variation indiameter and were often filled with dense granularmaterials. However, they contained no microtubules,
intermediate filaments or microfilaments. The tubular
projections were not found on the surface of intracystic
bodies (daughter cells) in the mature cysts. The struc‑ture of tubular projections and the mode of abhesion to

the type I epithelium was not different among rats,
nude mice and cats.

DISCUSSION
The pellicle of P. carinii trophozoites consists of a

25‑30 nm thick layer in which strata can be distin‑
guished: at the inner side a unit membrane as theboundary of the cytoplasm and on the outside a 20‑25
nm thick layer of moderately electron‑dense material.

asitism in the alveoli. The type I epithelium extends
and contracts when alveoli expand and contract withbreathing. Thus, it seems that the tubular projections
may help to fix the organisms to the motile type I
epithelium, especially when the number of organisms
had increased to fill the alveoli and many organisms had
contact with type I epitheliurn.
In the lightly infected lungs, gas diffusion throughthe respiratory membrane may be interfered with by themembrane to membrane attachment of P. carinii and bylimiting the functional area of the respiratory mem‑brane. Furthermore, the large tangles of tubular projec‑

tions, which were often found between the clusters oftrophozoites and respiratory membrane, may also inter‑fere with efficient ventilation locally.
In heavily infected lungs almost all alveoli are filledwith trophozoites, cysts, debris of the host cells andThis electron‑dense outer layer contains a considerable

occasionally with foamy macrophages (Shiota, 1984).

amount of carbohydrates whith bind to Concanavalin A
and a lectin from Macura ponrzfera (Yoshikawa et al.,In the present study, we found that many tubular projec‑
tions developed around the trophozoites and filled thealveoli. These projections seem to link the organisrns
together, creating a large mass of these organisms in the1988). These carbohydrates are known to play manyroles in cell to cell adhesion. In P. carinii cyst walls,

glucose, mannose and galactose were found and thesesugars may mediate P. carinii host interaction (DeStefano et al., 1990). A major surface glycoprotein (gp120) , which was of high‑mannose type, was detected inP. carinii, and a possible role for the adherence of P.
carinii through this glycoprotein to host mucin compo‑

nents in lung tissue was indicated by Radding et al(1989) .

Numerous reports have demonstrated that P. car‑alveoli anchored to the type I epithelial cells. Because

clearance of alveoli normally depends on macrophages
which are inhibited by immunosuppression, these masses

cannot be cleared. The mixture of organisms andmacrophages would need to advance into the bron‑chioles before becoming subject to removal by ciliary
action and coughing.

In conclusion, tubular projections may play animportant role in the adhesion of P. carinii to the typeinii attach thernselves to a specific alveolar lining cell,I epithelium and to one another preventing their being

the type I epithelium (Barton and Campbell, 1969;

cleared.

Vavra and Kucera, 1970; Ham et al., 1971; Lanken et al.,

1980; Yoneda and Walzer, 1980, 1981; Yoshida et al.,
1984; Henshaw et al., 1985). P. carinii is tightly
attached to the type I epithelium, as judged by freeze‑fracture electron microscopy (Yoneda and Walzer,
1983). The interaction of P. carinii with this cell playsa central role in the host‑parasite relationship in theinfection (Walzer, 1986) . Tubular projections hava beenknows for a long time (Yoneda and Walzer, 1980;

ACKNOWLEDGMENTS
We thank Emeritus Professor Dr. J. K. Frenkel,
Dept. of Pathology, Univ. of Kansas Medical Center,
Kansas City, Kansas, for his critical reading and com‑ments on the manuscript.
1 ) Barton, E. G. Jr. and Campbell, W. G. Jr. (1969): Pneumocys‑

Yoshida et al., 1984; Henshaw et al., 1985). P. carinii

tis carinii in lungs of rats treated with cortisone acetate:

can alter its morphology to fit the contour of its immedi‑

Ultrastructural observations relating to the life cycle, Amer.

ate surroundings. Indeed, the analogy of a rock clirnber
tenaciously grasping the side of a steep incline may wellreflect the situation with P. carinii (Long et al., 1986).

2 ) De Stefano, J. A., Cushion, M. T., Puvanesarajah, V. andWalzer P. D. (1990): Analysis of Pneumorystis carinii cyst

Some authors clearly show these structures, but do not
explain the process.
It seemed that P. carinii depends on the adherenceof the pellicles to the type I epithelium for its par‑J. Pathol., 54, 209‑236

wall. II. Sugar composition, J. Protozool., 37, 436‑441

3 ) Ebe, T., Sakashita, I. and Matsukawa, T. (1968): A new
method to fix lung tissues. J. Electron. Microsc., 17, 235‑236

4 ) Frenkel, J. K., Good, J. T. and Shultz, J. A. (1966): Latentpneumocystis infection of rats, relapse, and chemotherapy,

137
Lab. Invest., 15, 1559‑1577
5 ) Ham, E. K., Greenberg, S. D., Reynolds, R. C. and Singer, D.

B. (197D: Ultrastructure of Pneumorystis carinii, Exp.
Molec. Pathol., 14, 362‑372

6 ) Henshaw, N. G., Carson, J. L. and Collier, A. M. (1985):
Ultrastructural observations of Pneumo(ystis carinii attach‑ment to rat lung, J. Infect. Dis., 151, 181‑186

7 ) Lanken, P. N., Minda, M., Pietra, G. G. and Fishman, A. P.

(1980): Alveolar response to experimental Pneumo(ystis
carinii pneumonia in the rat, Amer. J. Pathol., 99, 561‑588
8 ) Long, E. G., Smith, J. S. and Meier, J. L. (1986): Attachmentof Pneumocystis carinii to rat pneumocyles, Lab. Invest., 54,

609‑6159 ) Radding, J. A., Armstrong, M. Y. K., Ullu, E. and Richards, F.
F. (1989): Identification and isolation of a major cell surface

glycoprotein of Pneumorystis carinii, Infect. Immun., 57,2149‑2157lO) Shiota, T. (1984): Morphology and development ofPneumorystis carinii observed by phase‑contrast microscopy
and semiultrathin section light‑microscopy, Jpn. J. Parasitol.,

133,433‑455

1D Shiota, T., Yamada, M. and Yoshida, Y. (1986): Morphology,
development and behavior of P leumorystis carinii observed
by light‑ microscopy in nude mice, Zbl. Bakt. Hyg., A262, 230‑23912) Shiota, T., Shimada, Y., Kurimoto, H.and Oikawa, H. (1990):
Pneumocystis carinii infection in corticosteroid‑treated cats,
J. Parasitol., 76, 441‑445

13) Takeuchi, S. (1980): Electronmicroscopic observation of
Pneumocystis carinii, Jpn. J. Parasitol., 29, 427‑453

14) Vavra, J. and Kucera, K. (1970): Pneumocystis carinii Der‑
an0 , its ultrastructure and ultrastructural affinities, J. Prot‑ozool., 17, 463‑483
15) Vossen, M. E. M. H., Beckers, P. J. A., Meuwissen, J. H. E. T.

and Stadhouders, A. M. (1978) : Developmental biology of
Pneumorystis carinii, an alternative view on the life cycle ofthe parasite, Ztschr. Parasitenkd., 55, 101‑118

16) Walzer, P. D. (1986) Attachment of microbes to host cells:
Relevance of Pneumorystis carinii, Lab. Invest., 54, 589‑592

17) Yoneda, K. and Walzer, P. D. (1980) : Interaction of
Pneumorystis carinii with host lungs: an ultrastructural
study, lpfect. Immun., 29, 692‑703

18) Yoneda, K. and Walzer, P. D. (198D: Mechanism of pulmo‑nary alveolar injury in experimental Pneumo(ystis carinii
pneumonia in the rat, Br. J. Exp. Pathol., 62, 339‑346

19) Yoneda, K. and Walzer, P. D. (1983): Attachment ofPneumocystis carinii to type I alveolar cells: studied byfreeze‑fracture electron microscopy, Infect. Immun., 40, 812‑81520) Yoshida, Y., Matsumoto, Y., Yamada, M., Okabayashi, K.,
Yoshikawa, H. and Sakazawa, M. (1984): Pneumocystis car‑inii: Electron microscopic investigation on the interaction oftrophozoite and alveolar lining cell, Zbl. Bakt. Hyg., A256,

390‑399
2D Yoshikawa, H., Morioka, H. and Yoshida, Y. (1987): Freeze‑fracture studies on Pneumocystis carinii. II. Fine structureof the trophozoite, Parasitol. Res., 73, 132‑139

22) Yoshikawa, H., Morioka, H. and Yoshida, Y. (1988) : Ultras‑

tructural detection of carbohydrates in the pellicle of
Pneumorystis carinii, Parasitol. Res., 74, 537‑543

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VOL22NO、3SEP

0『EMBER1994

CONTENTS
Original article
Yamauchi，S．，Ijichi，S．，Niina，K．，Hokezu，Y．，Maruyama，L，Niimura，T．and
OccultbyDiffuseMagnetic

Involvement

ResonanceofSupratentorial

Imagingin

HTLV−IWhite

in

Factors

Affecting

Prognosis

Osame7M

Detected

一117 120

Carriers一

Sow，P．S．，Diop，B．M．，Faye，M．A．，Badiane，S．，and

Epidemiological

MatterofColl−Seck，A．M

Neonatal

Tetanus一121−124

Dakar，Senegal．一

Nakas・ne7N
StoollwanagaンM・・Wasit・ン早・B・S・epart・・P・andGdeRanuh71・G・N・

Analysisof

Pediatric

DiarrheainSurabaya，Indonesia……

Higa，N．，Sithivong，N、and Iwanaga，M．、
A Comparative Study on S吻勿Jo6066爵側名召％s Isolated in Lao PDR and in Japan・・125−127

・・129−131

Shi。ta，T．，Shimada，Y．，Kurim。t。，H．，dikawa，H．andAriz・n・，N．
The

Roll

of

Tubular

Projections

of．P．6α7伽ガPneumonia…一of

P擁％彫o砂s廊6召万πガin

Pathogenicity
・・133−137、