Infectious agents, Leptospira spp. and Bartonella spp., in blood donors from Cajamarca, Peru

Introduction

The implementation of molecular and serological tests has led a great decline in transfusion-transmitted infections. Unfortunately, however, this has only occurred in high-income countries, whereas the scenario is different in low-income countries and in rural areas of middle-income countries, in which access to serological tests is sometimes not feasible or limited by economic factors^1,2. Furthermore, in middle and low-income countries, the burden of infectious diseases tends to be higher and subjects are often weakened by various factors, including malnutrition. These factors result in a population that is more vulnerable and at increased risk of infections. Although the search for relevant pathogens that can be transmitted by blood transfusion is implemented worldwide, various pathogens that can be present in blood bank donations remain under studied, as in the case of some bacteria such as Leptospira spp. and Bartonella spp. Bartonella species are re-emerging blood-borne organisms, capable of causing prolonged infections in animals and humans³, while leptospirosis is recognised as an emerging public health problem worldwide⁴. Both infections are considered to be neglected tropical diseases.

In humans, Bartonella spp. cause infections of varying severity, ranging from asymptomatic bacteraemia to severe cases with chronic manifestations responsible for persistent disease and death³. So far, various members of the Bartonella genus have been associated with human disease, the most relevant being Bartonella henselae, causing cat scratch disease, Bartonella quintana, causing trench fever, and Bartonella bacilliformis, the causal agent of Carrion’s disease². This last micro-organism is endemic in Latin America, and specifically in the Andean valleys. B. bacilliformis infects red blood cells and endothelial cells and, as mentioned above, is the aetiological agent of Carrion’s disease, a biphasic disease with two different clinical presentations: an initial febrile phase with haemolytic anaemia, known as Oroya fever, which has a mortality rate ranging from 44–88% in untreated patients, and a second phase characterised by the development of dermal eruptions, known as “Peruvian wart”⁵. It is important to highlight the presence of asymptomatic people, who account for about 45% of the population in some endemic areas⁶; consequently, the risk of transfusion transmission is of note, especially because of the ability of B. bacilliformis to survive in blood stored at 4 ºC for up to 1 year⁷.

Leptospirosis is one of the most common bacterial zoonotic diseases worldwide. Various mammals such as rodents, livestock and domestic pets can act as reservoir hosts⁸. Traditionally, infection in humans is due to contact with Leptospira-contaminated sites in different environments: at work (veterinarians, farmers, sewer cleaners), in alleys and slums with poor drainage, and in recent years, during international travel and recreational activities⁹. Infection by Leptospira may be asymptomatic or may cause a mild influenza-like illness that can resolve spontaneously. Symptomatic infection may present as non-specific symptoms such as fever, chills, headache, myalgia and jaundice⁸. As mentioned above, the presence of asymptomatic carriers may favour the risk of transfusion-related transmission. The distribution of Carrion’s disease is limited to the Andean valleys, while leptospirosis occurs worldwide, being more common in tropical and subtropical regions due to the wet weather. Rainfall, flooding and climatic phenomena, such as “El Niño”, increase the risk of these bacterial infections⁴.

There is no information about the prevalence of these bacteria in blood donors in Peru. Moreover, analyses to detect the presence of Leptospira spp. are not routine in this country and the presence of B. bacilliformis is only tested for in blood banks in endemic areas, but not in the remaining ones, irrespectively of whether there are inhabitants arriving from or travelling to endemic areas^10,11. This represents a health problem, since subjects receiving a blood transfusion are usually immunocompromised and more vulnerable to the development of severe infectious diseases from potentially transfusion-transmitted micro-organisms.

The aim of the present study was to examine the prevalence of Bartonella spp. and Leptospira spp. in blood donors in Cajamarca, in northern Peru, and describe the associated epidemiological factors.

Material and methods

Patients and sampling

The study was conducted from March to May of 2014 under a collaborative agreement between the Universidad Peruana de Ciencias Aplicadas (UPC) - Institute of Nutritional Studies (UPC-IIN, Lima, Peru) and the Blood Bank Service of the regional hospital of Cajamarca. This hospital is located in the capital of Cajamarca Department, in northern Peru, and serves an area greater than 35,000 km² and a population of approximately 1,500,000 people. The hospital’s catchment area has great climatic diversity: humid subtropical regions (eastern slopes), dry subtropical and tropical regions (western slopes) and, in the centre, a high mountain climate.

Healthy blood donors were recruited at the time of their volunteer blood donation at the Blood Bank of the regional hospital of Cajamarca. These blood donors were between 18–55 years of age, had no signs or symptoms of unspecified illness (no fever, chills, jaundice or myalgia in the preceding 4 weeks) and signed informed consent to participate in the study. All blood donors included in the study met at least one of the following inclusion criteria: exposure to water sources, water logging or other potentially contaminated water collections, such as irrigation canals, ditches, pools, ponds, lakes, and rivers; exposure to drains, latrines or management of wastewater contaminated with urine of rodents and other animals; people at occupational risk, such as farmers, ranchers, garbage collectors, recyclers, ditch cleaners, water and sewer workers, plumbers, veterinarians, agricultural technicians who treat animals and slaughterhouse workers; recreational and adventure sports that are related to potentially contaminated water sources (rivers, lakes, ditches, ponds and other) and living in rural and marginal urban housing with overcrowding or poor or absent sanitation.

Samples of 3 mL of venous blood from blood donors were collected into tubes containing EDTA and citrate, stored at 4 ºC and sent in on a weekly basis to the UPC-IIN (Lima, Peru) facilities where they were stored at 4 °C until processing. The project was approved by the Ethics Committees of the regional hospital of Cajamarca (Peru) and the Hospital Clinic of Barcelona (Spain).

DNA extraction

DNA was extracted from 200 μL of blood samples using a commercial extraction kit (High Pure Kit Preparation template, Roche Applied Science, Mannheim, Germany). Bacterial DNA obtained after extraction was eluted in 100 μL of nuclease free water and then processed or stored at −20 °C until use.

Amplification of a Bartonella spp.-specific 16S rRNA gene fragment

A 438 bp fragment of the 16S rRNA gene was amplified in blood samples as previously described¹² using the primers: p24Emod 5′-CCTTCAGTTMGGCTGGATC-3′ and 16S-R 5′-GCCYCCTTGCGGTTAGCACA-3′. Five microlitres of DNA extracted from blood were used in each polymerase chain reaction (PCR). The 438 bp amplified products were gel recovered, purified (SpinPrepTM Gel DNA Kit, San Diego, USA) and sent to be sequenced (Macrogen, Seoul, Korea).

Detection of Leptospira spp

Specific multiplex PCR able to detect all pathogenic Leptospira species described (L. interrogans, L. borgpetersenii, L. weilii, L. noguchii, L. santarosai, L. meyeri and L. kirschneri) was carried out using the primers G1 (5′-CTGAATCGCTGTATAAAAGT) and G2 (5′-GGAAAACAAATGGTCGGAAG), and primers B64-I (5′-CTGAATTCTCCATCTCAACTC) and B64-II (5′-GCAGAAATCAGATGGACGAT) as described previously¹³. In addition, 5 μL of DNA extracted from blood were used in each PCR. A positive sample, used as a control, was provided by the Regional Health Direction - DIRESA, Cajamarca.

Data analysis

Statistical significance was established using the Fisher’s exact test. Differences with a p-value <0.05 were considered statistically significant.

Results

During the study period, a total of 581 blood donors were received at the regional hospital of Cajamarca. Of these, 42 (7.23%) blood donors met at least one of the inclusion criteria and were consequently included in the analysis. Of the samples taken from these 42 blood donors, eight (19.05%) were positive for the presence of Leptospira spp. and one (2.38%) for Bartonella spp., which was classified as B. bacilliformis after sequencing (Figure 1).

Figure 1.

Flow chart of the presence of both Lesptospira and Bartonella spp. in blood donors at the regional hospital of Cajamarca.

PCR: polymerase chain reaction.

According to the demographic and epidemiological characteristics of the study population, 57.2% of the blood donors lived in rural areas and 59.5% reportedly worked as farmers and/or ranchers. Overall, 27 (64.3%) of the 42 blood donors studied were inhabitants of rural areas or farm/ranch workers living in urban areas, indicating continuous exposure to a rural environment. The remaining characteristics studied are shown in Table I. The only B. bacilliformis carrier lived in a rural area and worked as a farmer. Regarding the characteristics of Leptospira spp. carriers, five cases (62.5%) were reported to have been exposed to a rural environment. Four out of the eight (50%) Leptospira-positive donors lived in rural areas, working as farmers or ranchers, while the remaining lived in urban areas, although one was a farmer. Three cases were reported to have travelled to recognised Leptospira endemic areas in Cajamarca Department during the preceding 4 weeks (Table I). With regards to the possession of animals, 75% of Leptospira spp. carriers had a dog, while 50% had a cat. Regarding asymptomatic Leptospira carriers, 62.5% reported contact with backwaters and 100% had had contact with a septic pit latrine in the 4 weeks before blood donation. Meanwhile 62.5% had skin wounds and 75% reported contact with rodents during the preceding 4 weeks (Table I). None of the epidemiological factors analysed showed a statistically significant association with the presence of Leptospira (p-value ≥0.05).

Table I.

Demographic data of the blood donors studied.

		All donors (N=42) N (%)	Leptospira positive (n=8) n (%)	Bartonella positive (n=1) n (%)
Gender	Male	26 (62)	5 (62.5) (p=1.0)	0
	Female	16 (38)	3 (37.5) (p=1.0)	1
Age	Median (CI)	33.1 (29.8–36.4)	29.6 (22.1–37.1)	28
	18–30	23 (54.8)	6 (75)	1
	31–40	7 (16.6)	1 (12.5)	0
	41–50	8 (19.0)	0	0
	>50	4 (9.5)	1 (12.5)	0
Living area	Urban	18 (42.8)	4 (50) (p=1.0)	0
	Rural	24 (57.2)	4 (50) (p=1.0)	1
Overcrowding1		12 (28.6)	4 (50) (p=0.25)	0
Occupation2	Farmer/Rancher	25 (59.5)	4 (50) (p=0.71)	1
	Student	6 (14.3)	1 (12.5) (p=1.0)	0
	Health personnel	2 (4.8)	2 (25) (p=0.12)	0
	Others	9 (21.4)	1 (12.5) (p=1.0)	0
Travel3		7 (16.6)	3 (37.5) (p=0.33)	0
Animal contact4	Cattle	19 (45.2)	3 (37.5) (p=1.0)	1
	Pigs	12 (28.6)	2 (25) (p=1.0)	1
	Sheep	2 (4.8)	0 (p=1.0)	0
	Poultry	9 (21.4)	0 (p=0.32)	0
	Dogs	26 (61.9)	6 (75) (p=0.69)	0
	Cats	19 (45.2)	4 (50) (p=1.0)	1
Other	Backwaters5	19 (45.2)	5 (62.5) (p=0.46)	0
	Septic pit latrine5	32 (76.2)	8 (100) (p=0.18)	0
	Rodents5	22 (52.4)	6 (75) (p=0.44)	1
	Skin wounds6	17 (40.5)	5 (62.5) (p=0.28)	0

¹More than three people per bedroom;

²one farmer also reported being a student, while other four also reported other parallel activities;

³trip to a Leptospira endemic zone (in preceding 4 weeks);

⁴regular contact with livestock and pets;

⁵contact in the 4 weeks prior to donation;

⁶during the 4 weeks prior to donation.

CI: confidence interval.

Discussion

Infections by Leptospira spp. and Bartonella spp. in humans may be asymptomatic. The lack of evidence of carrier status may be a risk factor for blood donations, especially in areas in which these pathogens are not searched for. In the present study, we found evidence of Leptospira spp. and B. bacilliformis in 19% and 2.4%, respectively, of the samples from blood donors in Cajamarca. It is important to note that the percentage of samples positive for Bartonella may be underestimated, since studies have demonstrated that prior enrichment of the sample may increase positive results by 55% compared to those obtained when testing the original blood sample².

Although the authors of a study performed in an area with an increasing incidence of leptospirosis found no previous cases of Leptospira-infected transfusions in the region and no cases of acute infection in 485 blood donors studied, they did find evidence of past infections in some donors and proposed that acute bacteraemia in blood donors would provide the potential for transfusion-related transmission¹⁴. Nonetheless, only one case has been described in India¹⁵, demonstrating the possible presence of viable Leptospira in platelets¹⁶. With respect to Bartonella, blood transfusion is considered one of the mechanisms of transmitting B. bacilliformis infection, although to our knowledge it has only been described once¹⁷.

Leptospiraemia and bartonellosis occur mainly in rural and deprived urban areas, where they may exacerbate poverty by limiting the productivity of the population and impairing development, causing morbidity in these local populations¹⁸. These illnesses are not, however, limited to developing countries, being present also in developed countries. Indeed, a high prevalence of Bartonella spp. carriers was observed among the homeless¹⁹. In should be noted that all Bartonella spp. can be a risk for transfusions in blood banks, as reported recently². In blood banks, the presence of Leptospira, when sought for, is determined by serological techniques, while that of B. bacilliformis is determined by thin smear. The PCR method has shown good detection of Leptospira in blood donor screening, reducing the risk of infection by blood transfusion²⁰. As far as concerns Bartonella, although PCR analysis detects a higher number of infections than thin smear studies, the PCR technique seems to have limited value in detecting asymptomatic carriers²¹ because DNA amplification from original blood samples is relatively insensitive, primarily because the low level of bacteraemia typically present, especially in asymptomatic subjects, may be below the detection level of the PCR assay²¹. Given that asymptomatic leptospiraemia may not always be detected by routine bacterial culture methods available in rural hospitals, and that the diagnosis of bartonellosis remains a microbiological challenge because of the difficulty of culturing and isolating the causative agent from patients’ specimens²², these bacteria pose a risk to transfusion safety, especially in regions with a high prevalence of Leptospira/Bartonella. These high-risk regions are associated with climatic phenomena, such as flooding, concentrated in developing countries due to their intrinsic characteristics²². In addition, the lack of resources in these countries leads to less stringent screening of samples from blood donors and a consequent increase in the risk of infection by Leptospira or Bartonella^2,15,21. In the case of Peru, a middle income country, the national legislation related to blood banks requires that donations are tested for the presence of human immunodeficiency virus, human T-cell lymphotrophic viruses 1 and 2, hepatitis B virus, hepatitis C virus, Trypanosoma cruzii and Treponema pallidum, while detection of other pathogens such as B. bacilliformis or Plasmodium spp. is only considered in endemic areas^10,11. Thus Leptospira is not considered in screening, while the risk of a transfusion transmission of B. bacilliformis in blood banks within endemic areas is high. The blood bank of Cajamarca is in a B. bacilliformis-free urban area, although donors may live outside the city, as in the case of the carrier detected.

The presence of Leptospira spp. is usually related to excreta from humans or animals, including rodent urine²². Thus, risk factors for leptospirosis infection include skin cuts, abrasions, lack of footwear, and contact with contaminated water, soil or mud either during work or recreational activities²². The present study did not show a significant association between the risk factors considered and the presence of Leptospira spp. This may be related to the small sample size of the study, which is the study’s main limitation. However it should be considered that the majority of the factors studied are very common in Cajamarca inhabitants, thereby making it difficult to establish a relationship with leptospirosis.

Conclusions

Molecular tools should be routinely implemented in screening for Leptospira and Bartonella in blood banks, in order to diminish possible post-transfusion infections. The development of more sensitive diagnostic tools and evaluation of the adequacy of current blood service guidelines for the management of leptospirosis and bartonellosis in risk areas are needed.