Isolation frequency of medically important fungi and fluconazole resistant of Candida sp from hospital cockroaches.

Cockroaches are common pests in homes and hospitals. They are known to cause allergic reactions in certain individuals and have found to be potential vectors for various bacterial and parasitic pathogens. This study assessed the potential of hospital cockroaches to act as vectors of medically important fungal pathogens on their external surfaces. Cockroaches were captured from the main Intensive care unit (ICU), burn unit, adult surgical wards , pediatric oncology wards, intern hostel kitchen and the central kitchen of a national referral teaching hospital in Tanzania. Normal saline washings from the external surface of cockroaches were cultured on standard mycological media to facilitate isolation and identification of medically important molds and yeasts. Susceptibility of Candida sp isolates to fluconazole was tested using the CLSI M27-A3 microdilution method.


Conclusion
The external surfaces of cockroaches from this hospital may act as reservoirs of medically important opportunistic fungi exhibiting resistance to fluconazole.

Background
Cockroaches belong to the order Orthoptera or Dictyoptera and the families Blattidae or Blattelidae. They are common pests of domestic dwellings, hospitals and industrial areas.
They tend to live hide in the dark cracks and crevices of kitchens, toilets and food stores; as these are ideal environments in terms of temperature, humidity and sources of nutrition for them to flourish. Kitchens and toilets are areas that are often contaminated with infectious microorganisms including bacteria, viruses, protozoa and fungi, as result it may not be unexpected that cockroaches may be reservoirs of infectious microorganisms.
Nevertheless there are limited studies that have looked in role of cockroaches as vectors and reservoirs of opportunistic and HAI associated with fungal pathogens (9) Hospital care associated infections (HAI) are a major public health concern worldwide because of the morbidity, mortality and cost associated with them (10)(11). The WHO estimates that the prevalence of HAIs varies between 5.7% and 20.0% in low-and middle-income countries (12). Although elimination of HAIs is impossible, regular surveillance and effective infection control prevention (ICP) programs can help reduce its incidence. However, its implementation in developing countries is a challenge. Many developing country hospitals often lack adequate staff, equipment and supplies to effectively implement effective ICP measures. Basic infection control practices such as hand hygiene, are often not adhered too to the lack of clean running water (13). Moreover, the inability to regularly monitor and evaluate HAIs in these settings, may result in the delayed detection of outbreaks.
Despite these impediments it is imperative that health care institutions especially in resource limited settings strive to identify sources and implement preventative measures to the incidence of HAIs in their environments. Hospital environments provide the perfect environment in terms of temperature, humidity and sources of nutrition for them to flourish. In Tanzania, there is a limited of published data on prevalence of HAI and even less so data on the non-fomite vectors of fungal HAIs (17)(18)(19)(20). To our knowledge, this is the first report of a study performed in a Tanzanian hospital, investigation the role of cockroaches as vectors and reservoirs of HAI associated fungal infections.

Settings
The study to determine the prevalence of medically important fungi on te surface of hospital cockroaches was conducted at a national referral teaching Hospital located in Dar es Salaam, Tanzania. The facility is a 1,500 bed hospital, attending 1,000 to 1,200 outpatients per day, admitting 1,000 to 1,200 inpatients per week. The hospital has 25 departments and 106 units.

Study Design
An experimental laboratory based study was conducted . Hospital cockroaches were trapped, over a period of two months between February and March 2017. Cockroach adhesive Sticky paper ( Bbacb ®) was placed overnight on the floors of wards of the burn unit, intensive care unit , surgical department, neonatal unit, the hospital main kitchen, pediatric oncology and the students intern hostel kitchen at MNH. Trapped live 5 cockroaches were collected using sterile forceps and placed in sterile capped test tubes for transportation to the laboratory.

Fungal Isolation, Culture and identification
Sterile normal saline (10mls) was added to each test tube and the live cockroaches were thoroughly shaken for 2 minutes. Cockroach washings (5mls) was then aseptically removed to sterile tube for six set of 10 serial dilutions ranging from 1/10 to 1/10 6 of cockroach washings were then serially diluted in sterile water and 1 ml duplicate aliquots of the washings were cultured on Sabouraud's dextrose agar (Ponadisa) containing 0.5% chloramphenicol and gentamicin 0.05%. Plates were incubated at 30°C for 3 weeks and examined daily for viable counts. Filamentous fungi and yeast colonies were identified by using their microscopic and macroscopic characteristics, such as topography, texture, pigmentation, mycelium type, hyphae form, spore type and type of reproductive structure. Yeast were further identified by the germ tube test, the presence of chlamydo conidia on Corn meal plus Tween 80 agar (Oxoid, United Kingdom) and color of colonies on CHROMagar™ Candida .

Antifungal susceptibility profile of Candida Isolates
Susceptibility to fluconazole on 60 randomly selected Candida sp Isolates was tested using the CLSI M27-A3 microdilution method (21). Reference grade fluconazole was obtained powder from Sigma Aldrich, Germany. Antifungal susceptibility testing were performed in microdilution plates in which 0.1 mL of fluconazole 3X concentrate was used. The inocula were prepared from overnight (24-

Data processing and analysis
Laboratory data were entered and analysed using SPSS version 20. Descrptive statistics was presented as percentages in tables and figures

Results
A total of 72 cockroaches were captured from the different wards of the tertiary teaching hospital as shown in Figure 1. The majority of cockroaches were trapped from the Main hospital kitchen (15), followed by the Intern kitchen (14) Burn unit (9) and Neonatal unit (11), no cockroaches were captured from the intensive care unit.
Medically important Candida Sp. Including Candida albicans were isolated and identified ( 23%) as can be seen in Figure 4.
In addition to Candida sp., isolates of opportunistic pathogenic Aspergillus sp like Aspergillus fumigatus (50%) were also identified as can be seen in Figure 5.
Among the 108 randomly selected Candida isolates, 16.3% showed resistance to fluconazole. The majority of the resistant isolates found in isolates of C.parapsilosis and C. glabarata as shown in Table 1.

Discussion
Fungal HAIs infections continue to be a major cause of morbidity and mortality despite the current developments in terms of diagnosis and therapeutic options. This study is the first description of the fungal profile of the external surfaces of hospital cockroaches in a  (22)(23)(24)(25)(26)(27). While C. rugosa is now more and more being recognized as an emerging pathogen of blood stream infections of individuals who are immumocomprised (28).
The isolation of Aspergillus fumigatus and A. niger further supports the finding that hospital cockroaches may be potential reservoirs of major fungal pathogens associated with life threating HAIs infections of immunocompromised individuals. These isolates are known to be major causes of the invasive fungal infections particularly in transplant and Acquired immunodeficiency syndrome (AIDS) patients (25)(26). The presence of other known causes of opportunistic and nosocomial fungal infections species such as Cladosporium, Mucor, Fusarium , Rhizopus and Pencillum , further cements the concept that cockroaches in hospitals a may be major public health concern and confirm the findings of other studies conducted on domestic and hospital cockroaches (1)(2)(3)(4)(5)(6)(7)(8)(9) . What the study findings indicate is that there needs to be more studies to be conducted to ascertain if medically important fungi on the surfaces of hospital cockroaches are etiological agents of HAIs including of newer fungal pathogens immunocompromised patients such as Talaromyces marneffei (29).
The majority of cockroaches were trapped from the Main hospital kitchen and none cockroaches were captured from the main intensive care unit of this hospital. This indicates that infection prevention and control measures are being to adhered in sensitive hospital areas. However there needs to be concentrated efforts for effective fumigation practices in the food handling areas. Especially considering the fact, that cockroaches carrying drug resistant pathogens may wander from any part of the hospital through the hospital sewage systems unnoticed. Moreover , hospital environmental control teams therefore needs to do a thorough assessment of the resistance of the hospital cockroaches 9 to common pesticides before on deciding what chemicals to use. This is view of recent study findings that have shown that cockroaches in domestic and hospital settings are becoming resistant to common pesticides (30).
Fluconazole has been the corner stone therapeutic option for many African countries in treating fungal infections particularly for vaginal and oral candidiasis. This study found that 16.3% of randomly selected Candida isolates exhibited resistance to fluconazole. This is higher that has been previously reported in Tanzania were it shown that resistance to fluconazole was only 6.8% (30). This finding maybe a warning sign for the continued empirical use fluconazole for prophylaxis and or treatment of systemic or localized

Consent for publication
Not applicable

Availability of data and Materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Competing Interests
The authors declare no competing interest.

Funding
This was a non-funded project; the investing author used their own funds to support the data collection and analysis

Author Contribution
DM participated in fungal identification, antifungal susceptibility testing and drafted the manuscript. GB participated in antifungal susceptibility testing. KD conceived the study and drafted the manuscript. All authors read and approved the final manuscript.    Aspergillus sp. Isolation frequency on the external surfaces of cockroaches