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Keywords:

  • Cryptococcus neoformans;
  • chicken faeces;
  • Phayao;
  • Thailand;
  • HIV;
  • AIDS;
  • cryptococcal meningitis;
  • isolation

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

We successfully isolated Cryptococcus neoformans from chicken faeces in suburban areas of Thailand. C. neoformans was isolated from 36/150 houses (24.0%) in the dry season and 6/150 (4.0%) in the rainy season. All environmental isolates were of serotype A. The high isolation rate of 24% from chicken faeces has never been reported previously. Our environmental study could probably explain the high incidence of cryptococcal meningitis in HIV patients in Thailand. Copyright © 2004 John Wiley & Sons, Ltd.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Cryptococcal meningitis caused by Cryptococcus neoformans is one of the most serious opportunistic infections in patients with acquired immunodeficiency syndrome (AIDS), and also the gravest complication with respect to prognosis, especially in developing countries. A higher incidence of cryptococcal meningitis (18.5%) in patients with human immunodeficiency virus (HIV) infection has been described in Thailand compared to other developed countries. Especially, the percentage of AIDS and cryptococcosis in HIV-infected patients reported from the northern area of Thailand is larger than that from other areas (Suwat et al., 2001).

C. neoformans is known to inhabit natural environments such as soil and grows in bird excreta, especially that of pigeons (Ajello, 1958; Denton and DiSalvo, 1968; Yamamoto et al., 1995a, 1995b). C. neoformans has rarely been isolated from fruits, eucalyptus trees or other natural sources. The fungus spreads in the air and infects humans through inhalation. Although environmental studies have been performed, the source of C. neoformans in endemic areas of northern Thailand has never been elucidated.

As there is an office of the Japan International Cooperation Agency (JICA) in Phayao province, in northern Thailand, we were able to benefit from their cooperation. In Phayao province people breed many chickens under stilt houses and are exposed to potent aerosols of chicken faeces. To identify the source of C. neoformans in patients with cryptococcal meningitis, we focused on the layers of chicken faeces which are piled up under most houses or in the backyards of residential areas in Phayao province in which we could not find any pigeons, and determined whether they contained C. neoformans. Chiang Muan, Pon and Chiang Kham districts in Phayao were chosen for collection of faeces because the JICA office in Phayao were able to negotiate with medical staff there. We expected a higher isolation rate of C. neoformans from the dry faeces that can be collected in sunny periods. To our knowledge, there have been no studies comparing C. neoformans isolation rates in the dry and rainy seasons. In the present study, we compared the number of fungi that could be isolated from the chicken excreta in the dry month of March and the rainy month of December. Figure 1 shows climatic data for Chiang Rai in northern Thailand.

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Figure 1. Climatic data for Chiang Rai in northern Thailand

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Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Approximately 3.0–6.0 g weathered chicken excreta was added to 20 ml sterilized saline. The samples were allowed to soak for more than 1 h with frequent vortexing. The solution was filtered using sterilized nylon mesh with a pore size of 292 ± 10 µm (mean ± SEM) and was centrifuged at 3000 rpm for 10 min. The precipitates were inoculated onto birdseed agar plate, pH 6.8 ± 0.2 (BBL, MD). All plates were incubated at 30 °C for 2–6 days and a brown colony was streaked onto a Sabouraud dextrose agar, pH 5.6 ± 0.2 (Yamamoto et al., 1995b). All isolates were identified as C. neoformans by the Auxacolor system (Sanofi Diagnostics, Pasteur). Serotypes were confirmed by the Crypto check system (Iatron, Tokyo, Japan) (Ikeda et al., 1982; Kabasawa et al., 1991).

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

We collected chicken faeces from 90 dwellings in the Chiang Muan district, 30 in the Pon district and 30 in the Chiang Kham district, from under or around the private houses. In addition, 16 C. neoformans isolates from the cerebrospinal fluid of HIV patients were provided by Chiang Kham Hospital. Differences between groups were examined for statistical significance using the Fisher exact test. A p-value of <0.05 denoted the presence of a statistically significant difference.

C. neoformans was isolated from 36/150 (24.0%) houses in the dry season and 6/150 (4.0%) in the rainy season. All environmental isolates were serotype A (Table 1). Similarly, all strains from patients were of type A.

Table 1. Incidence of C. neoformans isolated from chicken excreta
 HousesVillagesSerotype
  • Data are number of isolates/total number examined (percentage).

  • *

    p < 0.05, compared with the rainy season.

Dry season36/150 (24%)*24/50 (48%)*All type A
Rainy season6/150 (4%)6/50 (12%)All type A

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Cryptococcosis is a deep-seated mycosis that can be classified roughly into three types of diseases; pulmonary cryptococcosis, cryptococcal meningitis and disseminated cryptococcosis. Cryptococcal meningitis is a fatal infection in patients with AIDS. As mentioned above, C. neoformans is prevalent in the natural environment, especially in the weathered excreta of birds. A high prevalence of C. neoformans in pigeon excreta has been well documented by numerous investigators in several parts of the world (Ajello, 1958; Denton et al., 1968; Yamamoto et al., 1995a, 1995b). Ajello (1958) isolated C. neoformans from both pigeon and chicken habitats. However, there are only a few reports of isolation of this organism from chicken habitats, and the isolation rate of C. neoformans from such environments is quite low. Walter and Yee (1968) attributed the growth inhibitory effect of chicken droppings on C. neoformans to the presence of a high molecular growth inhibitory substance and the high alkalinity of the droppings, which may explain the failure of isolation of the organism from chicken excreta. Also, Kielstein (1996) showed that the increase of pH is not regarded as responsible for the survival of C. neoformans in the birds' droppings.

In the Phayao province of northern Thailand, most people breed chickens in their yards. In the present study, we cultured aged and well-weathered faeces because birds faeces are not a primary source of C. neoformans (Monga et al., 1971; Littman and Walter, 1968). We succeeded in isolating C. neoformans from Phayao chicken excreta with a high isolation rate of 24% (36/150 houses) and showed an even higher isolation rate of 48% in 24/50 villages. Once C. neoformans is detected from one house, it is likely that there is C. neoformans in faeces from other houses in the same village. The isolation of C. neoformans is difficult because its growth on media may be affected by a variety of biotic factors, such as soil bacteria, amoebae, mites or sow bugs that are capable of inhibiting or killing the fungi. The weather in Thailand is considered appropriate for the growth of C. neoformans, with a mean temperature of 26–30 °C, and differs in rainy and dry seasons (Figure 1). Several studies have reported seasonal variation in isolation of C. neoformans from patients with AIDS (Bell et al., 2001; Bogaerts et al., 1999; Sorvillo et al., 1997). According to these reports, cryptococcosis in patients with AIDS is predominant in the rainy season, although our data showed the counterview. In cases where a patient inhales a lot of the pathogen in the dry season, the infection may become manifest after about a 6 month latency period. Thus, it is conceivable that the seasonal predominance of C. neoformans isolation is an important factor in cryptococcal infection. On the other hand, seasonal differences in the isolation rates do not necessarily account for the latency period of cryptococcosis. Considering a report of years-long latency, however, the most important factor that affects cryptococcal sideration is the host immune status. Endogenous reactivation of cryptococcosis, like tuberculosis, is also possible (Dromer et al., 1992; Blasi et al., 2001).

Generally, there is a high incidence of C. neoformans serotype B infection among the non-AIDS population in Thailand (Kwon-Chung and Bennett, 1984). However, our results showed that CSF isolates from AIDS patients were all of serotype A. These findings suggest that C. neoformans in the natural environment could cause cryptococcal meningitis. Yamamoto et al. (1995a) used strain-typing analysis by random amplified polymorphic DNA (RAPD) to confirm the genetic correlation between environmental isolates of C. neoformans and clinical isolates from patients with pulmonary cryptococcosis. In this regard, strain-typing studies are essential to determine the relationship between clinical and environmental isolates. Once the origin of infection is confirmed, this information could be useful for prevention of cryptococcal meningitis in such endemic areas as Phayao. In this study, although we tried to examine the relationship between environmental and clinical isolates of C. neoformans in Phayao province using RAPD analysis, RAPD did not reveal any clear relationships due to its poor resolution.

Our ultimate goal is to reduce the incidence of fatal cryptococcal infections. Further studies are needed to confirm our results in order to establish the importance of simple methods to prevent infection, such as effective removal or elimination of dry piled faeces.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The authors thank the Phayao Provincial Health Office, especially Dr Petcheri Sirinirund, Provincial Chief Medical Officer, the Region 10 Health Office and the Ministry of Public Health of the Kingdom of Thailand for their cooperation. The Japan International Cooperation Agency (JICA) and the JICA Project for Model Development of Comprehensive HIV/AIDS Prevention and Care provided technical support. The Ministry of Health, Labour and Welfare, Japan, provided technical and financial support. This work is partially supported by a grant from the Ministry of Health and Labour.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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