INTRODUCTION
Forty-one percent of the world’s population live in areas where malaria is transmitted, and an estimated 700,000–2.7 million persons die of malaria each year. (http://www.cdc.gov/malaria/facts.htm WorldMalaria.) In Japan, indigenous malaria was eradicated in 1961.1 Recently, an increasing number of Japanese have been traveling to malaria-endemic countries for business and vacation. People from countries with malaria endemicity have also been visiting Japan for education. This global travel has resulted in 100–160 cases of imported malaria per year.1–3 The number of physicians, however, who can diagnose malaria and treat it appropriately has fallen in Japan as the current priority of medical research and education has moved to chronic diseases such as cancer or cardiovascular diseases. Most technologists are also not trained to diagnose malaria on properly prepared thin and thick blood smears. Diagnostic delay with falciparum malaria can result in increased mortality, especially among nonimmune travelers. It is therefore important for physicians to have a low index of suspicion for malaria as a cause of acute febrile illness among Japanese travelers and immigrants from malaria-endemic countries.
Recently, two reports were published describing the global statistics of imported malaria in Japan based on data from the national surveillance system.1,2 To further evaluate clinical problems in the management of malaria in Japan, a detailed review of individual patient medical records is necessary. The Institute of Medical Science (IMS) Hospital at the University of Tokyo has been a referral center for cases of malaria in Japan for decades. Malaria cases managed at IMS Hospital account for 10–20% of the total cases in Japan. In this study, we reviewed patient medical records of 170 malaria cases seen at IMS Hospital from 1992 to 2001 and analyzed their demographic and clinical data.
MATERIALS AND METHODS
All confirmed cases of malaria at IMS Hospital from January 1992 to December 2001 were reviewed. The following parameters were included: age, sex, suspected area of contraction, nationality, Plasmodium species, antimalarial treatment, duration of symptoms prior to seeking medical care, use of chemoprophylaxis, complications, prognosis, and evidence of relapse or recrudescence. Patients who were treated abroad and visited IMS Hospital for follow-up after clearance of parasitemia were excluded unless their blood smears performed in Japan were positive. All cases were diagnosed with conventional microscopic examination of Giemsa staining of thin and thick blood films by technicians or physicians with expertise in tropical medicine, and the diagnosis was always confirmed by polymerase chain reaction (PCR) in indeterminant cases.4 Collected data were analyzed using EpiInfo2002 downloaded from the Web site of the U.S. Centers for Disease Control and Prevention. All statistical analyses were performed with two-tailed test, and P < 0.05 was considered statistically significant.
RESULTS
Overall, there were 170 confirmed cases of malaria at IMS Hospital from January 1992 to December 2001. There were no indigenously acquired cases. According to national surveillance systems, the annual number of malaria cases in Japan has remained stable at 103–156 cases for the decade.1,2 The number of cases at IMS Hospital has accounted for 9.0–21.4% (average 14.9%) of all cases in Japan. Seventeen of the 170 cases were relapses or recrudescence (4 episodes from 2 subjects with Plasmodium ovale, 12 episodes from 9 subjects with Plasmodium vivax, and 1 episode from a subject with Plasmodium falciparum) after treatment at IMS Hospital and were excluded from analysis to avoid redundancy. Of 153 cases, 101 were Japanese citizens and 52 were foreigners (Table 1). Seven of the 52 foreigners were from industrialized countries without indigenous malaria and had traveled to malaria endemic tropical countries prior to visiting Japan. The remaining 45 people were from malaria-endemic countries. There was no significant difference in demographic data between Japanese and foreign patients (Table 1). Plasmodium falciparum was the leading species among both Japanese and foreign patients, followed by Plasmodium vivax, Plasmodium ovale, and Plasmodium malariae (Table 1). The proportion of P. falciparum cases tended to increase between the first and second half of the decade. Nevertheless, there was no statistical significant difference (47.6% versus 58.6%, P = 0.176, χ2 test). Africa was the most frequent area of acquisition in both groups throughout the decade, followed by Oceania among Japanese patients and by Asia among foreign patients. Overall, Oceania overtook Asia as the second most common area of malaria acquisition in the second half of the decade (Asia versus Oceania: 21.7% versus 15.7% in the first half decade, 20.0% versus 24.3% in the second half decade).
P. falciparum infection accounted for 74.4% of cases from Africa, 40.0% from Oceania, 12.5% from Asia, and 0% from South America. These proportions remained stable through the decade (data not shown).
The purpose of travel to malaria-endemic areas was reported for all of Japanese patients with malaria. Contrary to our expectation, the proportion of travelers for sightseeing has not increased in the second half decade (first versus second half decade: 27.6% versus 27.9%), and business was the most common purpose through the decade (first versus second half decade: 70.7% versus 69.8%). On the other hand, visiting friends and relatives (VFR) accounted for only 1.0% in the decade.
The use of chemoprophylaxis was available for 100 of 101 Japanese patients. The percentage of Japanese patients with malaria who had taken chemoprophylaxis decreased dramatically between 1992–1996 and 1997–2001 (48.3 versus 14.3%, P = 0.0003). Although chloroquine was the leading agent used between 1992 and 1996 (Table 2), no patients took it from 1997 to 2001. Four patients acquired vivax malaria despite taking mefloquine for chemoprophylaxis. These four cases were thought to represent relapses, as none had taken primaquine after leaving the malarious areas.
Mefloquine has been the most frequently prescribed antimalarial treatment against P. falciparum infection (82.5%, data not shown). Chloroquine was used in several cases in the early 1990s but has not been used since 1997. Atovaquone/proguanil and artesunate have been used in a limited number of patients. Of 80 patients with falciparum malaria, only 4 cases had severe complications; 1 case of cerebral malaria, 2 cases of renal failure, and 1 case of severe anemia. There were, however, no deaths attributable to malaria. Of 38 cases of vivax malaria among Japanese patients, 36 were prescribed primaquine to prevent relapse after successful treatment with schizonticides. Despite primaquine prophylaxis, eight cases (22.2%) experienced relapse. Six of these cases (75.0%) contracted malaria in Oceania; mostly Papua New Guinea (Table 3). In cases in which relapse occurred after standard primaquine treatment (15 mg base per day for 14 days), modified regimens with larger doses or longer periods of primaquine were administered with favorable results (Table 3).
We analyzed the length from onset of symptoms to first medical consultation among Japanese patients (Figure 1). Data was available for 99% of Japanese patients. The mean and median duration was 4.7 days (95% confidence interval [CI]: 3.7–5.7) and 3.0 days (interquartile range [IQR]: 2.0–5.0 days), respectively. The mode was 2.0 days (18.9%). In cases of falciparum malaria, the mean and median duration were 4.1 days (95% CI: 2.5–5.7) and 2.0 days (IQR: 1.0–5.0), respectively. There were 12 falciparum malaria patients who visited clinics 5 days or more after onset of illness. Nine patients had taken chemoprophylaxis (chloroquine: 4; unknown: 1) and/or had self-administered antimalarials (chloroquine: 5; halofantrine: 1) prior to presentation. Of the three patients with delayed presentation who had not received antimalarials, one had renal failure (6 days) and the other two had no complications (7 and 11 days).
DISCUSSION
This retrospective review of malaria cases at a national referral hospital corresponds to 14.9% of the total cases in Japan over 10 years. This study represents the largest review of clinical characteristics and outcomes of patients with malaria in Japan. Falciparum malaria accounted for about 50% of total malaria cases at the IMS Hospital. The proportion of P. falciparum cases is relatively high in France (around 80%), moderate in Germany and the United Kingdom (around60%), and low in the United States (around 40%).5 The proportion of falciparum malaria cases in Japan seems to lie between European countries and the United States. European countries are geographically close to Africa where P. falciparum is a dominant species, whereas the United States is closer to Central and South America where P. vivax is predominant. Japanese travel to both Africa and Asia/Oceania likely accounts for this midrange proportion of P. falciparum in IMS Hospital.
VFR, which is one of the most common reasons for travel to malarious areas in Western countries, accounted for only 1.0% of Japanese travelers. In the United Kingdom, where immigrants compose a substantial percentage of populations, VFR, holidays, and business accounted for 56%, 12%, and 6.5%, respectively.6 Because Japan is racially homogeneous, VFR is not likely to be a major reason for travel to malaria-endemic countries. Business travels accounted for 70% among Japanese patients throughout the decade. In Germany, where the number of immigrants from malarious countries is small like Japan, however, business travel accounted for only 18%, and 75% was holiday travels.7 This contrasting result might be explained by the difference in the number of sightseeing travelers to malarious areas between Japan and Germany. Nevertheless, detailed investigation would be required to clarify it. Improving travel advisements to overseas employees is likely to contribute to reduction in the number of imported malaria in Japan.
There are no national guidelines for malarial prophylaxis or treatment in Japan. At IMS Hospital, mefloquine is used to treat falciparum malaria without complications, intravenous quinine for severe falciparum malaria, and chloroquine for nonfalciparum malaria. Although we have not experienced mefloquine-resistant falciparum malaria, it is well-known that multidrug-resistant falciparum malaria has emerged in Southeast Asia, especially at the border between Thailand and Myanmar and between Thailand and Cambodia.8,9 Because many Japanese visit Thailand, health care providers must be aware of potential resistance when treating patients returning from these areas. Fortunately, we experienced no deaths from malaria at our institution. However, we previously reported that the case fatality rate (CFR) nationally from falciparum malaria is 3.3%.2 This CFR is as high as that of Germany (3.6%) and much higher than that of France (1.98%), the United States (1.01%), and the United Kingdom (0.65%).5 A high CFR could be attributable to better mortality reporting compared with total case reporting,5 a high proportion of patients without immunity to malaria,10 or poor management of complicated malaria.10 These factors may explain the discrepancy of CFR between the IMS Hospital and the rest of Japan. Alternatively, travelers who are aware of their malaria risk may present earlier to reference hospitals for tropical medicine. Unawareness of malarial risk will lead to delayed infectious diseases physician consultation and result in unfavorable outcomes.
P. vivax infection generally causes nonfatal disease. Primaquine administration after treatment with schizonticides is required to eradicate the dormant form of P. vivax in the liver. Because primaquine is not always effective against hypnozoites, relapse is occasionally observed even after adequate primaqine therapy.11,12 Of the cases of vivax malaria that relapsed after primaquine therapy at IMS Hospital, 75% were from Oceania. Both primaquine and chloroquine resistant P. vivax have emerged in the same area.13 We have shown that the number of malaria cases from Oceania has increased from 1997 to 2001. And further increases are likely, as Japanese travel to Papua New Guinea is increasing. We, therefore, are more likely to encounter imported cases of primaquine and/or chloroquine resistant P. vivax. Cases of vivax malaria from Oceania, therefore, require careful observation during treatment of the acute febrile phase and consideration for a modified dosage or duration of primaquine (for example, a longer duration or higher dose of primaquine therapy).14
Although there had been neither guidelines nor approved drugs for malaria chemoprophylaxis in Japan until 2001, travelers used to collect information and get drugs in a variety of ways. We unexpectedly found that the proportion of Japanese patients who were taking chemoprophylaxis had dropped drastically in 1997–2001. The difference may reflect the absence of chloroquine failures, as this drug was used less frequently for prophylaxis. This suggests that Japanese travelers are correctly informed of chemoprophylactic regimens that successfully prevent malaria acquisition. In support of this speculation, the annual number of imported malaria cases at the IMS Hospital and nationally has remained stable in the past decade despite record high levels of travel by both Japanese people and foreigners in 2000 (see http://www.immi-moj.go.jp/toukei/index.html). However, given the substantial number of travelers who still contract malaria without chemoprophylaxis, further efforts must be made to educate travelers.
The interval between the onset of symptoms and presentation to a hospital is another concern because any delay in diagnosis can lead to increased mortality with falciparum malaria. Kain and others reported that the mean duration from onset of symptoms until first medical consultation in Canadian travelers was 3.6 days (95% CI: 2.5–4.7) in hospitals without expertise in tropical medicine and 3.8 days (95% CI: 2.3–5.3) in hospitals with a tropical medicine unit.15 The mean duration in our cases was 4.7 days (95% CI: 3.7–5.7). However, because the duration in our cases was not normally distributed (Figure 1), a precise comparison with our data is difficult. Nevertheless, even a median duration of 3.0 days in our cases is an unacceptable length that could cause severe malaria or death in a nonimmune population. Delayed diagnosis despite early presentation to hospitals is another common problem that can increase mortality of falciparum malaria at health care facilities lacking an infectious diseases unit. However, because almost all cases in the IMS Hospital were diagnosed on the first day of presentation, the data shown above also represents the duration from the onset of symptoms to diagnosis.
This retrospective study reveals clinical problems relevant to malaria imported to Japan. A high frequency of relapse of vivax malaria despite primaquine administration in patients from Oceania must be relayed to health care providers. In addition, the absence of chemoprophylaxis and the delay in initial medical consultation suggest a continued ignorance of travelers regarding malarial risk. To reduce morbidity and mortality due to malaria, travelers must be informed of malaria risks, the necessity of chemoprophylaxis, and the importance of immediate medical consultation if fever develops. To this end, it is important to develop travel medicine referral centers to coordinate the education of health care providers and travel companies regarding malaria.
Characteristics of imported malaria in the past decade in IMS hospital
| All | Japanese | Foreigner | P value | |
|---|---|---|---|---|
| * Excluding relapse and recrudescence after treatment of initial event. | ||||
| † Student t test. | ||||
| ‡ χ2 test. | ||||
| § Pf, Pv, Po, and Pm represent P. falciparum, P. vivax, P. ovale, and P. malariae, respectively. Pf/Pv means mixed infection of Pf and Pv. | ||||
| ¶ “Africa/Asia” and “Asia/Oceania” mean that patients traveled around more than one area, and it was impossible to determine the estimated contraction area. | ||||
| Number of all cases | 170 | 116 | 54 | – |
| Number of new cases* | 153 | 101 | 52 | – |
| Age (mean ± SD) | 34.8 ± 11.2 | 35.4 ± 12.7 | 33.8 ± 7.7 | 0.42† |
| Male:female | 124:29 | 84:17 | 40:12 | 0.35‡ |
| Species N (%)§ | ||||
| Pf | 80 (52.3) | 50 (49.5) | 30 (57.7) | |
| Pv | 55 (35.9) | 38 (37.6) | 17 (32.7) | |
| Po | 14 (9.2) | 11 (10.9) | 3 (5.8) | 0.46‡ |
| Pm | 3 (2.0) | 1 (1.0) | 2 (3.8) | |
| Pf/Pv | 1 (0.7) | 1 (1.0) | 0 (0.0) | |
| Total | 153 (100) | 101 (100) | 52 (100) | – |
| Suspected contraction areas N (%)¶ | ||||
| Africa | 83 (54.2) | 51 (50.5) | 32 (61.5) | |
| Asia | 32 (20.9) | 19 (18.8) | 13 (25.0) | |
| Oceania | 30 (19.6) | 25 (24.8) | 5 (9.6) | |
| South America | 3 (2.0) | 1 (1.0) | 2 (3.8) | 0.15‡ |
| Africa/Asia | 3 (2.0) | 3 (3.0) | 0 (0.0) | |
| Asia/Oceania | 1 (0.7) | 1 (1.0) | 0 (0.0) | |
| EU | 1 (0.7) | 1 (1.0) | 0 (0.0) | |
| Total | 153 (100) | 101 (100) | 52 (100) | – |
Comparison of chemoprophylaxis between 1992–1996 and 1997–2001
| Chemoprophylaxis among Japanese patients N (%) | 1992–1996 | 1997–2001 | P value* |
|---|---|---|---|
| *χ2 test between 1992–1996 and 1997–2001. | |||
| CP, chloroquine/proguanil; SP, sulfadoxine/pyrimethamine. | |||
| Chloroquine | 18 (31.0) | 0 (0.0) | 0.0006 |
| CP | 2 (3.4) | 0 (0.0) | |
| Mefloquine | 1 (1.7) | 3 (7.1) | |
| SP | 1 (1.7) | 0 (0.0) | |
| Others | 6 (10.3) | 3 (7.1) | |
| None | 30 (51.7) | 36 (85.7) | |
| Total | 58 (100) | 42 (100) | – |
List of vivax malaria cases that had relapses after primaquine treatment
| Case | Sex | Age | Year of first episode | Contracted countries | Episode | Schizonticides | Primaquine regimens |
|---|---|---|---|---|---|---|---|
| CRQ, chloroquine; MAL, atovaquone/proguanil. | |||||||
| 1 | M | 30 | 1992 | Malaysia | 1st | CRQ | 15 mg base for 14 days |
| 2nd | CRQ | 30 mg base for 7 days | |||||
| 3rd | CRQ | 15 mg base for 14 days × 2 courses | |||||
| 2 | M | 23 | 1993 | Papua New Guinea | 1st | CRQ | 15 mg base for 14 days |
| 2nd | CRQ | 15 mg base for 14 days × 2 courses | |||||
| 3 | M | 44 | 1993 | Papua New Guinea, Indonesia | 1st | CRQ | 15 mg base for 14 days |
| 2nd | CRQ | 15 mg base for 14 days | |||||
| 3rd | CRQ | 15 mg base for 14 days × 2 courses | |||||
| 4 | M | 22 | 1997 | Papua New Guinea | 1st | CRQ | 15 mg base for 14 days |
| 2nd | CRQ | 15 mg base for 14 days × 2 courses | |||||
| 3rd | CRQ | 30 mg for 11 days | |||||
| 5 | M | 32 | 1997 | Papua New Guinea, Solomon Island | 1st | MEF | 15 mg base for 9 days |
| 2nd | CRQ | Lost to follow-up | |||||
| 6 | M | 34 | 1998 | Philippines | 1st | CRQ | 15 mg base for 14 days |
| 2nd | CRQ | 15 mg base for 14 days | |||||
| 7 | M | 49 | 2001 | Papua New Guinea | 1st | CRQ | 15 mg base for 14 days |
| 2nd | CRQ | 30 mg base for 14 dyas | |||||
| 3rd | CRQ | 30 mg base for 28 days | |||||
| 8 | M | 24 | 2001 | Vanuatu | 1st | CRQ | 15 mg base for 14 days |
| 2nd | MAL | 30 mg base for 14 days | |||||
Distribution of period from the onset of illness to first medical consultation. Horizontal axis represents days from onset of symptoms to first medical consultation of malaria patients. Longitudinal axis indicates the number of cases. The bar represents total number of cases, and the black portion in each bar corresponds to the cases of falciparum malaria.
Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 73, 3; 10.4269/ajtmh.2005.73.599
Distribution of period from the onset of illness to first medical consultation. Horizontal axis represents days from onset of symptoms to first medical consultation of malaria patients. Longitudinal axis indicates the number of cases. The bar represents total number of cases, and the black portion in each bar corresponds to the cases of falciparum malaria.
Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 73, 3; 10.4269/ajtmh.2005.73.599
Distribution of period from the onset of illness to first medical consultation. Horizontal axis represents days from onset of symptoms to first medical consultation of malaria patients. Longitudinal axis indicates the number of cases. The bar represents total number of cases, and the black portion in each bar corresponds to the cases of falciparum malaria.
Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 73, 3; 10.4269/ajtmh.2005.73.599
Address correspondence to Tetsuya Nakamura, Department of Infectious Diseases and Applied Immunology, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai Minato-ku, Tokyo 108-8639, Japan. E-mail: tnakamur@ims.u-tokyo.ac.jp
Authors’ addresses: Toshiyuki Miura, Tokiomi Endo, Hitomi Nakamura, Takashi Odawara, and Aikichi Iwamoto, Division of Infectious Diseases, Advanced Clinical Research Center, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai Minato-ku, Tokyo 108-8639 Japan, Telephone: +81-3-5449-5338, Fax: +81-3-5449-5427. Tomohiko Koibuchi and Tetsuya Nakamura, Department of Infectious Diseases and Applied Immunology, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai Minato-ku, Tokyo 108-8639, Japan, Telephone: +81-3-5449-5338, Fax: +81-3-5449-5427. Mikio Kimura, Infectious Disease Surveillance Center, National Institute of Infectious Diseases, 1-23-1 Toyama Shinjuku-ku, Tokyo 162-8640, Japan, Telephone: +81-3-5285-1111, Fax: +81-3-5285-1129. Yusuke Wataya, Department of Drug Informatics, Faculty of Pharmaceutical Sciences, Okayama University, 1-1-1 Tsushima-naka, Okayama 700-8530, Japan, Telephone: +81-86-251-7976, Fax: +81-86-251-7974.
Acknowledgments: The authors thank Dr. Jay Keystone (Division of Infectious Diseases, Department of Medicine, University of Toronto) for his advice for the treatment of primaquine-resistant vivax malaria. We also thank Dr. Philip Peters (Division of Infectious Diseases, Emory University) for reviewing the language of the manuscript.
Financial support: This work was sponsored by the research grant for “Research on Health Sciences Focusing on Drug Innovation” from the Japan Health Sciences Foundation and Health and Labor Science Research Grants from the Ministry of Health, Labor and Welfare in Japan.
REFERENCES
- 2↑
Kimura M, Suzaki A, Matsumoto Y, Nakajima K, Wataya Y, Ohtomo H, 2003. Epidemiological and clinical aspects of malaria in Japan. J Travel Med 10 :122–127.
- 3↑
Ohtomo H, Takeutchi T, 1998. Studies on current trend of imported malaria in Japan. Jpn J Trop Med Hyg 26 :151–156.
- 4↑
Kimura M, Miyake H, Kim HS, Tanabe M, Arai M, Kawai S, Yamane A, Wataya Y, 1995. Species-specific PCR detection of malaria parasites by microtiter plate hybridization: clinical study with malaria patients. J Clin Microbiol 33 :2342–2346.
- 5↑
Muentener P, Schlagenhauf P, Steffen R, 1999. Imported malaria (1985–95): trends and perspectives. Bull World Health Organ 77 :560–566.
- 6↑
Bradley DJ, Warhurst DC, Blaze M, Smith V, Williams J, 1998. Malaria imported into the United Kingdom in 1996. Euro Surveill 3 :40–42.
- 8↑
Nosten F, ter Kuile F, Chongsuphajaisiddhi T, Luxemburger C, Webster HK, Edstein M, Phaipun L, Thew KL, White NJ, 1991. Mefloquine-resistant falciparum malaria on the Thai-Burmese border. Lancet 337 :1140–1143.
- 9↑
Smithuis FM, van Woensel JB, Nordlander E, Vantha WS, ter Kuile FO, 1993. Comparison of two mefloquine regimens for treatment of Plasmodium falciparum malaria on the northeastern Thai-Cambodian border. Antimicrob Agents Chemother 37 :1977–1981.
- 10↑
Jelinek T, Schulte C, Behrens R, Grobusch MP, Coulaud JP, Bisoffi Z, Matteelli A, Clerinx J, Corachan M, Puente S, Gjorup I, Harms G, Kollaritsch H, Kotlowski A, Bjorkmann A, Delmont JP, Knobloch J, Nielsen LN, Cuadros J, Hatz C, Beran J, Schmid ML, Schulze M, Lopez-Velez R, Fleischer K, Kapaun A, McWhinney P, Kern P, Atougia J, Fry G, da Cunha S, Boecken G, 2002. Imported falciparum malaria in Europe: sentinel surveillance data from the European network on surveillance of imported infectious diseases. Clin Infect Dis 34 :572–576.
- 11↑
Kimura M, Tomizawa I, Takizawa Y, Ohtomo H, 1996. A study of relapsed cases of vivax malaria after standard primaquine therapy. Kansenshogaku Zasshi 70 :1086–1091.
- 12↑
Jelinek T, Nothdurft HD, Von Sonnenburg F, Loscher T, 1995. Long-term efficacy of primaquine in the treatment of vivax malaria in nonimmune travelers. Am J Trop Med Hyg 52 :322–324.
- 13↑
Schuurkamp GJ, Spicer PE, Kereu RK, Bulungol PK, Rieckmann KH, 1992. Chloroquine-resistant Plasmodium vivax in Papua New Guinea. Trans R Soc Trop Med Hyg 86 :121–122.
- 14↑
Hatz C, 2001. Clinical treatment of malaria in returned travelers. Schlagenhauf P, ed. Travelers’ Malaria. Hamilton: BC Decker Inc., 431–445.
- 15↑
Kain KC, Harrington MA, Tennyson S, Keystone JS, 1998. Imported malaria: prospective analysis of problems in diagnosis and management. Clin Infect Dis 27 :142–149.